Combination therapy comprising anti-angiogenesis agents and ox40 binding agonists

ABSTRACT

The invention provides compositions and methods for treating cancers. The method comprises administering an anti-angiogenesis agent and an OX40 binding agonist.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication Ser. No. 61/973,193, filed Mar. 31, 2014; 61/989,448, filedMay 6, 2014; 62/073,873, filed Oct. 31, 2014; 62/080,171, filed Nov. 14,2014; and 62/113,345, filed Feb. 6, 2015; each of which is incorporatedherein by reference in its entirety.

SEQUENCE LISTING

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 146392031540SEQLIST.txt,date recorded: Mar. 26, 2015, size: 188 KB).

FIELD OF THE INVENTION

This invention relates to methods of treating cancers by administeringan anti-angiogenesis agent and an OX40 binding agonist.

BACKGROUND

Angiogenesis is necessary for cancer development, regulating not onlyprimary tumor size and growth, but also impacting invasive andmetastatic potential. Accordingly, the mechanisms mediating angiogenicprocesses have been investigated as potential targets for directedanti-cancer therapies. Early in the study of angiogenic modulators, thevascular endothelial growth factor (VEGF) signaling pathway wasdiscovered to regulate angiogenic activity in multiple cancer types, andmultiple therapeutics have been developed to modulate this pathway atvarious points. Although the use of angiogenesis inhibitors in theclinic has shown success, not all patients respond or fully respond tothis therapy. The mechanism(s) underlying such incomplete response isunknown. Therefore, there is a need for the identification of patientsubgroups sensitive or responsive to anti-angiogenic cancer therapy.Further, there remains a need for combination therapies that mayincrease the efficacy of anti-angiogenic cancer therapy.

Bevacizumab (Avastin®) is a recombinant humanized monoclonal IgG1antibody that specifically binds to and blocks the biological effects ofVEGF. Bevacizumab has been approved in Europe for the treatment of theadvanced stages of six common types of cancer: colorectal cancer, breastcancer, non-small cell lung cancer (NSCLC), ovarian cancer, cervicalcancer, and kidney cancer, which collectively cause over 2.5 milliondeaths each year. In the United States, bevacizumab was the firstanti-angiogenesis therapy approved by the FDA, and it is now approvedfor the treatment of six tumor types: colorectal cancer, NSCLC, braincancer (glioblastoma), kidney cancer (renal cell carcinoma), ovariancancer, and cervical cancer. Over half a million patients have beentreated with bevacizumab so far, and a comprehensive clinical program isinvestigating the further use of bevacizumab in the treatment ofmultiple cancer types.

Bevacizumab has shown promise as a co-therapeutic, demonstratingefficacy when combined with a broad range of chemotherapies and otheranti-cancer treatments. For example, phase-III studies have demonstratedthe beneficial effects of combining bevacizumab with standardchemotherapeutic regimens (see, e.g., Saltz et al., 2008, J. Clin.Oncol., 26:2013-2019; Yang et al., 2008, Clin. Cancer Res.,14:5893-5899; Hurwitz et al., 2004, N. Engl. J. Med., 350:2335-2342).However, as in previous studies of angiogenesis inhibitors, some ofthese phase-III studies have shown that a portion of patients experienceincomplete response to the addition of bevacizumab to theirchemotherapeutic regimens. Accordingly, there is a need for methods ofidentifying those patients that are likely to respond or have animproved response to not only angiogenesis inhibitors (e.g.,bevacizumab) alone, but also combination therapies comprisingangiogenesis inhibitors (e.g., bevacizumab).

Accordingly, there is a need for combination therapies that may increasethe efficacy of anti-angiogenic cancer therapy. Combination therapiesmay increase responsiveness in patients that show incomplete responseand/or further increase responsiveness in patients that do respond toanti-angiogenic cancer therapy.

OX40 (also known as CD34, TNFRSF4 and ACT35) is a member of the tumornecrosis factor receptor superfamily. OX40 is not constitutivelyexpressed on naïve T cells, but is induced after engagement of the Tcell receptor (TCR). The ligand for OX40, OX40L, is predominantlyexpressed on antigen presenting cells. OX40 is highly expressed byactivated CD4+ T cells, activated CD8+ T cells, memory T cells, andregulatory T cells. OX40 signaling can provide costimulatory signals toCD4 and CD8 T cells, leading to enhanced cell proliferation, survival,effector function and migration. OX40 signaling also enhances memory Tcell development and function.

Regulatory T cells (Treg) cells are highly enriched in tumors and tumordraining lymph nodes derived from multiple cancer indications, includingmelanoma, NSCLC, renal, ovarian, colon, pancreatic, hepatocellular, andbreast cancer. In a subset of these indications, increased intratumoralT reg cell densities are associated with poor patient prognosis,suggesting that these cells play an important role in suppressingantitumor immunity. OX40 positive tumor infiltrating lymphocytes havebeen described.

Modulating OX40 signaling with other signaling pathways that arederegulated in tumor cells (e.g., angiogenic pathways) may furtherenhance treatment efficacy. Thus, there remains a need for such anoptimal therapy for treating or delaying development of various cancers,immune related diseases, and T cell dysfunctional disorders.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY

In one aspect, provided herein is a method for treating or delayingprogression of cancer in an individual comprising administering to theindividual an effective amount of an anti-angiogenesis agent and an OX40binding agonist.

In another aspect, provided herein is a use of an anti-angiogenesisagent in the manufacture of a medicament for treating or delayingprogression of cancer in an individual, wherein the medicament comprisesthe anti-angiogenesis agent and an optional pharmaceutically acceptablecarrier, and wherein the treatment comprises administration of themedicament in combination with a composition comprising an OX40 bindingagonist and an optional pharmaceutically acceptable carrier. Furtherprovided herein is a use of an OX40 binding agonist in the manufactureof a medicament for treating or delaying progression of cancer in anindividual, wherein the medicament comprises the OX40 binding agonistand an optional pharmaceutically acceptable carrier, and wherein thetreatment comprises administration of the medicament in combination witha composition comprising an anti-angiogenesis agent and an optionalpharmaceutically acceptable carrier.

In still another aspect, provided herein is a composition comprising ananti-angiogenesis agent and an optional pharmaceutically acceptablecarrier for use in treating or delaying progression of cancer in anindividual, wherein the treatment comprises administration of saidcomposition in combination with a second composition, wherein the secondcomposition comprises OX40 binding agonist and an optionalpharmaceutically acceptable carrier. Further provided herein is acomposition comprising an OX40 binding agonist and an optionalpharmaceutically acceptable carrier for use in treating or delayingprogression of cancer in an individual, wherein the treatment comprisesadministration of said composition in combination with a secondcomposition, wherein the second composition comprises ananti-angiogenesis agent and an optional pharmaceutically acceptablecarrier.

In yet another aspect, provided herein is a kit comprising a medicamentcomprising an anti-angiogenesis agent and an optional pharmaceuticallyacceptable carrier, and a package insert comprising instructions foradministration of the medicament in combination with a compositioncomprising an OX40 binding agonist and an optional pharmaceuticallyacceptable carrier for treating or delaying progression of cancer in anindividual. Further provided here is a kit comprising a first medicamentcomprising an anti-angiogenesis agent and an optional pharmaceuticallyacceptable carrier, and a second medicament comprising an OX40 bindingagonist and an optional pharmaceutically acceptable carrier. In someembodiments, the kit further comprises a package insert comprisinginstructions for administration of the first medicament and the secondmedicament for treating or delaying progression of cancer in anindividual. Still further provided herein is a kit comprising amedicament comprising an OX40 binding agonist and an optionalpharmaceutically acceptable carrier, and a package insert comprisinginstructions for administration of the medicament in combination with acomposition comprising an anti-angiogenesis agent and an optionalpharmaceutically acceptable carrier for treating or delaying progressionof cancer in an individual.

In some embodiments, the anti-angiogenesis agent is selected from thegroup consisting of an anti-VEGFR2 antibody; an anti-VEGFR1 antibody; aVEGF-trap; a bispecific VEGF antibody; a bispecific antibody comprisinga combination of two arms selected from the group consisting of ananti-VEGF arm, an anti-VEGFR1 arm, and an anti-VEGFR2 arm; ananti-VEGF-A antibody; an anti-VEGFB antibody; an anti-VEGFC antibody; ananti-VEGFD antibody; a nonpeptide small molecule VEGF antagonist; ananti-PDGFR inhibitor; and a native angiogenesis inhibitor. In someembodiments, the anti-angiogenesis agent is selected from the groupconsisting of ramucirumab, tanibirumab, aflibercept, icrucumab,ziv-aflibercept, MP-0250, vanucizumab, sevacizumab, VGX-100, pazopanib,axitinib, vandetanib, stivarga, cabozantinib, lenvatinib, nintedanib,orantinib, telatinib, dovitinig, cediranib, motesanib, sulfatinib,apatinib, foretinib, famitinib, imatinib, and tivozanib.

In some embodiments, the anti-angiogenesis agent is an anti-angiogenesisantibody. In some embodiments, the anti-angiogenesis antibody is amonoclonal antibody. In some embodiments, the anti-angiogenesis antibodyis a human or humanized antibody. In some embodiments, theanti-angiogenesis agent is a VEGF antagonist. In some embodiments, theVEGF antagonist reduces the expression level or biological activity ofVEGF by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In someembodiments, the VEGF is VEGF (8-109), VEGF (1-109), or VEGF₁₆₅. In someembodiments, the VEGF antagonist increases MHC class II expression ondendritic cells as compared to MHC class II expression on dendriticcells prior to treatment with the VEGF antagonist. In some embodiments,the VEGF antagonist increases OX40L expression on dendritic cells ascompared to OX40L expression on dendritic cells prior to treatment withthe VEGF antagonist. In some embodiments, the dendritic cells aremyeloid dendritic cells. In some embodiments, the dendritic cells arenon-myeloid dendritic cells. In some embodiments, the VEGF antagonistcomprises a soluble VEGF receptor or a soluble VEGF receptor fragmentthat specifically binds to VEGF. In some embodiments, the VEGFantagonist is a chimeric VEGF receptor protein. In some embodiments, theVEGF antagonist is administered by gene therapy.

In some embodiments, the VEGF antagonist is an anti-VEGF antibody. Insome embodiments, the anti-VEGF antibody is a human or humanizedantibody. In some embodiments, the anti-VEGF antibody binds to theA4.6.1 epitope. In some embodiments, the anti-VEGF antibody binds to afunctional epitope comprising residues F17, M18, D19, Y21, Y25, Q89,191, K101, E103, and C104 of human VEGF. In some embodiments, theanti-VEGF antibody binds to a functional epitope comprising residuesF17, Y21, Q22, Y25, D63, 183, and Q89 of human VEGF. In someembodiments, the anti-VEGF antibody is a G6 series antibody. In someembodiments, the anti-VEGF antibody is a B20 series antibody. In someembodiments, the anti-VEGF antibody is a monoclonal anti-VEGF antibody.In some embodiments, the monoclonal anti-VEGF antibody is bevacizumab.In some embodiments, the anti-VEGF antibody comprises a light chainvariable region comprising the amino acid sequence of DIQMTQSPSSLSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTDFTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKR. (SEQ ID NO:214). In someembodiments, the anti-VEGF antibody comprises a heavy chain variableregion comprising the amino acid sequence of EVQLVESGGG LVQPGGSLRLSCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAYLQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS (SEQ ID NO:215). In someembodiments, the anti-VEGF antibody comprises a light chain variableregion comprising the amino acid sequence of DIQMTQSPSS LSASVGDRVTITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ YSTVPWTFGQ GTKVEIKR. (SEQ ID NO:214) and a heavy chainvariable region comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTFSLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS (SEQ IDNO:215). In some embodiments, the anti-VEGF antibody comprises one, two,three, four, five, or six hypervariable region (HVR) sequences ofbevacizumab. In some embodiments, the anti-VEGF antibody comprises one,two, three, four, five, or six hypervariable region (HVR) sequences ofselected from (a) HVR-H1 comprising the amino acid sequence ofGYTFTNYGMN (SEQ ID NO:216); (b) HVR-H2 comprising the amino acidsequence of WINTYTGEPTYAADFKR (SEQ ID NO:217); (c) HVR-H3 comprising theamino acid sequence of YPHYYGSSHWYFDV (SEQ ID NO:218); (d) HVR-L1comprising the amino acid sequence of SASQDISNYLN (SEQ ID NO:219); (e)HVR-L2 comprising the amino acid sequence of FTSSLHS (SEQ ID NO:220);and (f) HVR-L3 comprising the amino acid sequence of QQYSTVPWT (SEQ IDNO:221). In some embodiments, the anti-VEGF antibody comprises one, two,three, four, five, or six hypervariable region (HVR) sequences of anantibody described in U.S. Pat. No. 6,884,879. In some embodiments, theanti-VEGF antibody comprises one, two, or three hypervariable region(HVR) sequences of a light chain variable region comprising thefollowing amino acid sequence: DIQMTQSPSS LSASVGDRVT ITCSASQDISNYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQYSTVPWTFGQ GTKVEIKR. (SEQ ID NO:214) and/or one, two, or threehypervariable region (HVR) sequences of a heavy chain variable regioncomprising the following amino acid sequence: EVQLVESGGG LVQPGGSLRLSCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAYLQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS (SEQ ID NO:215). In someembodiments, the anti-VEGF antibody comprises one, two, three, four,five, or six hypervariable region (HVR) sequences of bevacizumab.

In some embodiments, the OX40 binding agonist is selected from the groupconsisting of an OX40 agonist antibody, an OX40L agonist fragment, anOX40 oligomeric receptor, and an OX40 immunoadhesin. In someembodiments, the OX40 binding agonist is a trimeric OX40L-Fc protein. Insome embodiments, the OX40 binding agonist is an OX40L agonist fragmentcomprising one or more extracellular domains of OX40L. In someembodiments, the OX40 binding agonist is an OX40 agonist antibody thatbinds human OX40. In some embodiments, the OX40 agonist antibodydepletes cells that express human OX40. In some embodiments, the OX40agonist antibody depletes cells that express human OX40 in vitro. Insome embodiments, the cells are CD4+ effector T cells. In someembodiments, the cells are Treg cells. In some embodiments, thedepleting is by ADCC and/or phagocytosis. In some embodiments, thedepleting is by ADCC. In some embodiments, the OX40 agonist antibodybinds human OX40 with an affinity of less than or equal to about 1 nM.In some embodiments, the OX40 agonist antibody depletes cells thatexpress human OX40 in vitro and binds human OX40 with an affinity ofless than or equal to about 1 nM. In some embodiments, the OX40 agonistantibody binds human OX40 with an affinity of less than or equal toabout 0.45 nM. In some embodiments, the OX40 agonist antibody bindshuman OX40 with an affinity of less than or equal to about 0.4 nM. Insome embodiments, OX40 agonist antibody binding affinity is determinedusing radioimmunoassay. In some embodiments, binding to human OX40 hasan EC50 of less than or equal to 0.2 ug/ml. In some embodiments, bindingto human OX40 has an EC50 of less than or equal to 0.3 ug/ml. In someembodiments, the OX40 agonist antibody increases CD4+ effector T cellproliferation and/or increasing cytokine production by the CD4+ effectorT cell as compared to proliferation and/or cytokine production prior totreatment with anti-human OX40 agonist antibody. In some embodiments,the cytokine is gamma interferon. In some embodiments, the OX40 agonistantibody increases memory T cell proliferation and/or increasingcytokine production by the memory cell. In some embodiments, thecytokine is gamma interferon. In some embodiments, the OX40 agonistantibody inhibits Treg function. In some embodiments, the OX40 agonistantibody inhibits Treg suppression of effector T cell function. In someembodiments, effector T cell function is effector T cell proliferationand/or cytokine production. In some embodiments, the effector T cell isa CD4+ effector T cell. In some embodiments, the OX40 agonist antibodyincreases OX40 signal transduction in a target cell that expresses OX40.In some embodiments, OX40 signal transduction is detected by monitoringNFkB downstream signaling. In some embodiments, the OX40 agonistantibody is stable after treatment at 40° C. for two weeks. In someembodiments, the OX40 agonist antibody comprises a variant IgG1 Fcpolypeptide comprising a mutation that eliminates binding to humaneffector cells, and wherein the antibody has diminished activityrelative to an anti-human OX40 agonist antibody comprising a nativesequence IgG1 Fc portion. In some embodiments, the OX40 agonist antibodycomprises a variant Fc portion comprising a DANA mutation. In someembodiments, OX40 agonist antibody cross-linking is required foranti-human OX40 agonist antibody function. In some embodiments, the OX40agonist antibody comprises (a) a VH domain comprising (i) HVR-H1comprising the amino acid sequence of SEQ ID NO: 2, 8 or 9, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO: 3, 10, 11, 12, 13 or14, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQID NO: 4, 15, or 19; and (iv) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:5, (v) HVR-L2 comprising the amino acid sequence of SEQ IDNO:6, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7, 22, 23, 24, 25, 26, 27, or 28. In some embodiments, the OX40 agonistantibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising anamino acid sequence of SEQ ID NO:7. In some embodiments, the OX40agonist antibody comprises (a) HVR-H1 comprising the amino acid sequenceof SEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3comprising an amino acid sequence of SEQ ID NO:26. In some embodiments,the OX40 agonist antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:3; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO:4; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e)HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3comprising an amino acid sequence of SEQ ID NO:27. In some embodiments,the OX40 agonist antibody comprises a heavy chain variable domain (VH)sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO:56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 108, 114, 116, 233, or 234. In someembodiments, the OX40 agonist antibody comprises a light chain variabledomain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO:57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 109, 115 or 117. In some embodiments, the OX40agonist antibody comprises a heavy chain variable domain (VH) sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to the amino acid sequence of SEQ ID NO:56. Insome embodiments, the OX40 agonist VH sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-human OX40agonist antibody comprising that sequence retains the ability to bind tohuman OX40. In some embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO:56. In someembodiments, the OX40 agonist VH comprises one, two or three HVRsselected from: (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:2, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4. In someembodiments, the OX40 agonist antibody comprises a light chain variabledomain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO:57. In some embodiments, the OX40 agonist VL sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-human OX40agonist antibody comprising that sequence retains the ability to bind tohuman OX40. In some embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO: 57. In someembodiments, the OX40 agonist VL comprises one, two or three HVRsselected from (a) HVR-L1 comprising the amino acid sequence of SEQ IDNO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In someembodiments, the OX40 agonist antibody comprises a VH sequence of SEQ IDNO: 56. In some embodiments, the OX40 agonist antibody comprises a VLsequence of SEQ ID NO: 57. In some embodiments, the OX40 agonistantibody comprises a VH sequence of SEQ ID NO:56 and a VL sequence ofSEQ ID NO: 57. In some embodiments, the OX40 agonist antibody comprisesa VH sequence of SEQ ID NO: 94. In some embodiments, the OX40 agonistantibody comprises a VL sequence of SEQ ID NO: 95. In some embodiments,the OX40 agonist antibody comprises a VH sequence of SEQ ID NO:94 and aVL sequence of SEQ ID NO: 95. In some embodiments, the OX40 agonistantibody comprises a VH sequence of SEQ ID NO: 96. In some embodiments,the OX40 agonist antibody comprises a VL sequence of SEQ ID NO: 97. Insome embodiments, the OX40 agonist antibody comprises a VH sequence ofSEQ ID NO:96 and a VL sequence of SEQ ID NO: 97. In some embodiments,the OX40 agonist antibody is MEDI6469, MEDI0562, or MEDI6383.

In some embodiments, the OX40 agonist is in a pharmaceutical formulationthat comprises any of the anti-human OX40 antibodies (e.g., agonistantibodies) provided herein and a pharmaceutically acceptable carrier.In some embodiments, the pharmaceutical formulation comprises (a) any ofthe anti-human OX40 agonist antibodies described herein at aconcentration between about 10 mg/mL and about 100 mg/mL, (b) apolysorbate, wherein the polysorbate concentration is about 0.02% toabout 0.06%; (c) a histidine buffer at about pH 5.0 to about pH 6.0; and(d) a saccharide, wherein the saccharide concentration is about 120 mMto about 320 mM. In some embodiments, the histidine buffer is at pH 5.0to 6.0. In some embodiments, the saccharide is sucrose. In someembodiments, the pharmaceutical formulation comprises (a) any of theanti-human OX40 agonist antibodies described herein, (b) polysorbate 20,wherein the polysorbate concentration is about 0.02%; (c) a histidineacetate buffer at pH 6.0; and (d) sucrose, wherein the sucroseconcentration is about 320 mM. In some embodiments, the pharmaceuticalformulation comprises (a) any of the anti-human OX40 agonist antibodiesdescribed herein, (b) polysorbate 20, wherein the polysorbateconcentration is about 0.02%; (c) a histidine acetate buffer at pH 5.5;and (d) sucrose, wherein the sucrose concentration is about 240 mM. Insome embodiments, the pharmaceutical formulation comprises (a) any ofthe anti-human OX40 agonist antibodies described herein, (b) polysorbate20, wherein the polysorbate concentration is about 0.04%; (c) ahistidine acetate buffer at pH 6.0; and (d) sucrose, wherein the sucroseconcentration is about 120 mM. In some embodiments, the pharmaceuticalformulation comprises (a) any of the anti-human OX40 agonist antibodiesdescribed herein, (b) polysorbate 40, wherein the polysorbateconcentration is about 0.04%; (c) a histidine acetate buffer at pH 5.0;and (d) sucrose, wherein the sucrose concentration is about 240 mM. Insome embodiments, the pharmaceutical formulation comprises (a) any ofthe anti-human OX40 agonist antibodies described herein, (b) polysorbate40, wherein the polysorbate concentration is about 0.04%; (c) ahistidine acetate buffer at pH 6.0; and (d) sucrose, wherein the sucroseconcentration is about 120 mM. In some embodiments, the antibody of theformulation comprises (a) a VH domain comprising (i) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 2, 8 or 9, (ii) HVR-H2 comprisingthe amino acid sequence of SEQ ID NO: 3, 10, 11, 12, 13 or 14, and (iii)HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 4, 15,or 19; and (iv) HVR-L1 comprising the amino acid sequence of SEQ IDNO:5, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 7, 22, 23,24, 25, 26, 27, or 28. In some embodiments, the antibody of theformulation comprises (a) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:7. In someembodiments, the antibody of the formulation comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprisingthe amino acid sequence of SEQ ID NO:3; (c) HVR-H3 comprising the aminoacid sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acidsequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequenceof SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid sequenceselected from SEQ ID NO:26. In some embodiments, the antibody of theformulation comprises (a) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:27. In someembodiments, the antibody of the formulation comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO:56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 108, 114, 116, 183, or 184.In some embodiments, the antibody of the formulation comprises a lightchain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID NO:57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 109, 115 or 117. In some embodiments,the antibody of the formulation comprises a heavy chain variable domain(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO:56. In some embodiments, the VH sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-human OX40agonist antibody comprising that sequence retains the ability to bind tohuman OX40. In some embodiments, total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO:56. In someembodiments, the VH comprises one, two or three HVRs selected from: (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:4. In some embodiments,the antibody of the formulation comprises a light chain variable domain(VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to the amino acid sequence of SEQ ID NO:57. Insome embodiments, the VL sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-human OX40 agonist antibody comprisingthat sequence retains the ability to bind to human OX40. In someembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 57. In some embodiments, the VLcomprises one, two or three HVRs selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino acidsequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7. In some embodiments, the antibody of theformulation comprises a VH sequence of SEQ ID NO: 56. In someembodiments, the antibody of the formulation comprises a VL sequence ofSEQ ID NO: 57. In some embodiments, the antibody of the formulationcomprises a VH sequence of SEQ ID NO:56 and a VL sequence of SEQ ID NO:57. In some embodiments, the antibody of the formulation comprises a VHsequence of SEQ ID NO: 94. In some embodiments, the antibody of theformulation comprises a VL sequence of SEQ ID NO: 95. In someembodiments, the antibody of the formulation comprises a VH sequence ofSEQ ID NO:94 and a VL sequence of SEQ ID NO: 95. In some embodiments,the antibody of the formulation comprises a VH sequence of SEQ ID NO:96. In some embodiments, the antibody of the formulation comprises a VLsequence of SEQ ID NO: 97. In some embodiments, the antibody of theformulation comprises a VH sequence of SEQ ID NO:96 and a VL sequence ofSEQ ID NO: 97. In some embodiments, the antibody of the formulationcomprises a VH sequence of SEQ ID NO: 179. In some embodiments, theantibody of the formulation comprises a VL sequence of SEQ ID NO: 180.In some embodiments, the antibody of the formulation comprises a VHsequence of SEQ ID NO: 179 and a VL sequence of SEQ ID NO: 180. In someembodiments, the antibody of the formulation comprises a VH sequence ofSEQ ID NO: 181. In some embodiments, the antibody of the formulationcomprises a VL sequence of SEQ ID NO: 182. In some embodiments, theantibody of the formulation comprises a VH sequence of SEQ ID NO: 181and a VL sequence of SEQ ID NO: 182.

In some embodiments, the cancer is lung cancer, glioblastoma, cervicalcancer, ovarian cancer, breast cancer, colon cancer, colorectal cancer,fallopian tube cancer, peritoneal cancer, kidney cancer, renal cancer,non-Hodgkins lymphoma, prostate cancer, pancreatic cancer, soft-tissuesarcoma, kaposi's sarcoma, carcinoid carcinoma, head and neck cancer,mesothelioma, multiple myeloma, non-small cell lung cancer,neuroblastoma, melanoma, gastric cancer, or liver cancer. In someembodiments, the cancer is a gynecologic cancer. In some embodiments,the cancer is advanced, refractory, recurrent, chemotherapy-resistantand/or platinum-resistant. In some embodiments, the individual hascancer or has been diagnosed with cancer. In some embodiments, thetreatment results in a sustained response in the individual aftercessation of the treatment. In some embodiments, the OX40 bindingagonist is administered before the anti-angiogenesis agent, simultaneouswith the anti-angiogenesis agent, or after the anti-angiogenesis agent.In some embodiments, the individual is a human. In some embodiments, theanti-angiogenesis agent and/or the OX40 binding agonist are administeredintravenously, intramuscularly, subcutaneously, intracerobrospinally,topically, orally, transdermally, intraperitoneally, intraorbitally, byimplantation, by inhalation, intrathecally, intraventricularly,intra-articularly, intrasynov ially, or intranasally. In someembodiments, the method further comprises administering achemotherapeutic agent for treating or delaying progression of cancer.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art. These andother embodiments of the invention are further described by the detaileddescription that follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the efficacy of different treatments on inhibiting tumorgrowth in a CT26 tumor model. Average tumor volumes (y-axis) over time(x-axis) are plotted for each experimental group. Experimental groupswere anti-OX40 and anti-GP120 treatment (pluses), anti-GP120 treatment(circles), anti-VEGF and anti-GP120 treatment (triangles), and anti-VEGFand anti-OX40 treatment (X's).

FIGS. 2A-2D track tumor volumes from individual mice over time in thefollowing treatment groups: anti-GP120 (control; FIG. 2A),anti-VEGF+anti-GP120 (FIG. 2B), anti-OX40+anti-GP120 (FIG. 2C), andanti-VEGF+anti-OX40 (FIG. 2D). Solid black and dashed and dotted linesrepresent tumors from individual mice within each experimental group.Solid black lines represent mice that remained alive at the terminationof the experiment, and dashed and dotted lines represent mice that wereeuthanized prior to experiment termination due to tumor ulceration ortumor size exceeding 2000 mm³. Evenly dashed lines depict the averagetumor volume over time in mice that received anti-GP120 alone (aslabeled by arrows). Unevenly dashed lines are representative of theaverage tumor volume over time within each experimental group (aslabeled by arrows). Percentages in top left corner of each individualgraph are % tumor growth inhibition (TGI), as judged against mice thatreceived anti-GP120 alone.

FIGS. 3A & 3B show increased intratumoral dendritic cell activationfollowing anti-VEGF treatment in a CT26 tumor model. FIG. 3A showsincreased activation of myeloid dendritic cells (CD11b+). FIG. 3B showsincreased activation of non-myeloid dendritic cells (CD11b−). Asterisksindicate statistical significance determined using a Student's t-test,assuming unequal variance and a significance level of 0.05 (* indicatesp<0.05).

DETAILED DESCRIPTION

In one aspect, provided herein are methods, compositions and uses fortreating or delaying progression of cancer in an individual comprisingadministering an effective amount of an anti-angiogenesis agent and anOX40 binding agonist.

I. Definitions

The term “dysfunction” in the context of immune dysfunction, refers to astate of reduced immune responsiveness to antigenic stimulation.

The term “dysfunctional”, as used herein, also includes refractory orunresponsive to antigen recognition, specifically, impaired capacity totranslate antigen recognition into downstream T-cell effector functions,such as proliferation, cytokine production (e.g., gamma interferon)and/or target cell killing.

“Enhancing T cell function” means to induce, cause or stimulate aneffector or memory T cell to have a renewed, sustained or amplifiedbiological function. Examples of enhancing T-cell function include:increased secretion of γ-interferon from CD8⁺ effector T cells,increased secretion of γ-interferon from CD4+ memory and/or effectorT-cells, increased proliferation of CD4+ effector and/or memory T cells,increased proliferation of CD8+ effector T-cells, increased antigenresponsiveness (e.g., clearance), relative to such levels before theintervention. In one embodiment, the level of enhancement is at least50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. Themanner of measuring this enhancement is known to one of ordinary skillin the art.

“Tumor immunity” refers to the process in which tumors evade immunerecognition and clearance. Thus, as a therapeutic concept, tumorimmunity is “treated” when such evasion is attenuated, and the tumorsare recognized and attacked by the immune system. Examples of tumorrecognition include tumor binding, tumor shrinkage and tumor clearance.

“Immunogenicity” refers to the ability of a particular substance toprovoke an immune response. Tumors are immunogenic and enhancing tumorimmunogenicity aids in the clearance of the tumor cells by the immuneresponse.

“Sustained response” refers to the sustained effect on reducing tumorgrowth after cessation of a treatment. For example, the tumor size mayremain to be the same or smaller as compared to the size at thebeginning of the administration phase. In some embodiments, thesustained response has a duration at least the same as the treatmentduration, at least 1.5×, 2.0×, 2.5×, or 3.0× length of the treatmentduration.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “agonist antibody,” as used herein, is an antibody which activates abiological activity of the antigen it binds.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted immunoglobulin bound onto Fcreceptors (FcRs) present on certain cytotoxic cells (e.g. NK cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The primary cells for mediatingADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI,FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarizedin Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92(1991). To assess ADCC activity of a molecule of interest, an in vitroADCC assay, such as that described in U.S. Pat. No. 5,500,362 or5,821,337 or U.S. Pat. No. 6,737,056 (Presta), may be performed. Usefuleffector cells for such assays include PBMC and NK cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in an animal model such as that disclosed inClynes et al. PNAS (USA) 95:652-656 (1998). An exemplary assay forassessing ADCC activity is provided in the examples herein.

The terms “anti-OX40 antibody” and “an antibody that binds to OX40”refer to an antibody that is capable of binding OX40 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting OX40. In one embodiment, the extent ofbinding of an anti-OX40 antibody to an unrelated, non-OX40 protein isless than about 10% of the binding of the antibody to OX40 as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to OX40 has a dissociation constant (Kd) of ≤1 μM, ≤100 nM,≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less,e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certainembodiments, an anti-OX40 antibody binds to an epitope of OX40 that isconserved among OX40 from different species.

As use herein, the term “binds”, “specifically binds to” or is “specificfor” refers to measurable and reproducible interactions such as bindingbetween a target and an antibody, which is determinative of the presenceof the target in the presence of a heterogeneous population of moleculesincluding biological molecules. For example, an antibody that binds toor specifically binds to a target (which can be an epitope) is anantibody that binds this target with greater affinity, avidity, morereadily, and/or with greater duration than it binds to other targets. Inone embodiment, the extent of binding of an antibody to an unrelatedtarget is less than about 10% of the binding of the antibody to thetarget as measured, e.g., by a radioimmunoassay (RIA). In certainembodiments, an antibody that specifically binds to a target has adissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM.In certain embodiments, an antibody specifically binds to an epitope ona protein that is conserved among the protein from different species. Inanother embodiment, specific binding can include, but does not requireexclusive binding.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “binding domain” refers to the region of a polypeptide thatbinds to another molecule. In the case of an FcR, the binding domain cancomprise a portion of a polypeptide chain thereof (e.g. the alpha chainthereof) which is responsible for binding an Fc region. One usefulbinding domain is the extracellular domain of an FcR alpha chain.

A polypeptide with a variant IgG Fc with “altered” FcR, ADCC orphagocytosis activity is one which has either enhanced or diminished FcRbinding activity (e.g, FcγR) and/or ADCC activity and/or phagocytosisactivity compared to a parent polypeptide or to a polypeptide comprisinga native sequence Fc region.

The term “OX40,” as used herein, refers to any native OX40 from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed OX40 as well as any form of OX40that results from processing in the cell. The term also encompassesnaturally occurring variants of OX40, e.g., splice variants or allelicvariants. The amino acid sequence of an exemplary human OX40 (minus thesignal peptide) is as shown in SEQ ID NO:1.

“OX40 activation” refers to activation, of the OX40 receptor. Generally,OX40 activation results in signal transduction.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Included in this definition are benign andmalignant cancers. Examples of cancer include but are not limited to,carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularexamples of such cancers include epithelial ovarian cancer, fallopiantube cancer, primary peritoneal cancer, squamous cell cancer, lungcancer (including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer), pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer (including platinum sensitive andplatinum resistant ovarian cancer), liver cancer, bladder cancer,hepatoma, neuroblastoma, melanoma, breast cancer, colon cancer,colorectal cancer, fallopian tube, peritoneal, endometrial or uterinecarcinoma, gynecologic cancers (e.g., ovarian, peritoneal, fallopiantube, cervical, endometrial, vaginal, and vulvar cancer), salivary glandcarcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulvalcancer, thyroid cancer, soft-tissue sarcoma, kaposi's sarcoma, carcinoidcarcinoma, mesothelioma, multiple myeloma, hepatic carcinoma and varioustypes of bead and neck cancer, as well as B-cell lymphoma (including lowgradeifollicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL)NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL;high grade immunoblastic NHL; high grade lymphoblastic NHL; high gradesmall non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chroniclymphocy tic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass),which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed. Polypeptidevariants with altered Fc region amino acid sequences (polypeptides witha variant Fc region) and increased or decreased C1q binding capabilityare described, e.g., in U.S. Pat. No. 6,194,551 B1 and WO 1999/51642.See also, e.g., Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The term “cytostatic agent” refers to a compound or composition whicharrests growth of a cell either in vitro or in vivo. Thus, a cytostaticagent may be one which significantly reduces the percentage of cells inS phase. Further examples of cytostatic agents include agents that blockcell cycle progression by inducing GO/G1 arrest or M-phase arrest. Thehumanized anti-Her2 antibody trastuzumab (HERCEPTIN®) is an example of acytostatic agent that induces GO/G1 arrest. Classical M-phase blockersinclude the vincas (vincristine and vinblastine), taxanes, andtopoisomerase II inhibitors such as doxorubicin, epirubicin,daunorubicin, etoposide, and bleomycin. Certain agents that arrest G1also spill over into S-phase arrest, for example, DNA alkylating agentssuch as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,methotrexate, 5-fluorouracil, and ara-C. Further information can befound in Mendelsohn and Israel, eds., The Molecular Basis of Cancer,Chapter 1, entitled “Cell cycle regulation, oncogenes, andantineoplastic drugs” by Murakami et al. (W.B. Saunders, Philadelphia,1995), e.g., p. 13. The taxanes (paclitaxel and docetaxel) areanticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®,Rhone-Poulenc Rorer), derived from the European yew, is a semisyntheticanalogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel anddocetaxel promote the assembly of microtubules from tubulin dimers andstabilize microtubules by preventing depolymerization, which results inthe inhibition of mitosis in cells.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

A “depleting anti-OX40 antibody,” is an anti-OX40 antibody that kills ordepletes OX40-expressing cells. Depletion of OX40 expressing cells canbe achieved by various mechanisms, such as antibody-dependentcell-mediated cytotoxicity and/or phagocytosis. Depletion ofOX40-expressing cells may be assayed in vitro, and exemplary methods forin vitro ADCC and phagocytosis assays are provided herein. In someembodiments, the OX40-expressing cell is a human CD4+ effector T cell.In some embodiments, the OX40-expressing cell is a transgenic BT474 cellthat expresses human OX40.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” is at least the minimum amount required to effecta measurable improvement or prevention of a particular disorder. Aneffective amount herein may vary according to factors such as thedisease state, age, sex, and weight of the patient, and the ability ofthe antibody to elicit a desired response in the individual. Aneffective amount is also one in which any toxic or detrimental effectsof the treatment are outweighed by the therapeutically beneficialeffects. For prophylactic use, beneficial or desired results includeresults such as eliminating or reducing the risk, lessening theseverity, or delaying the onset of the disease, including biochemical,histological and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. For therapeutic use, beneficial or desiredresults include clinical results such as decreasing one or more symptomsresulting from the disease, increasing the quality of life of thosesuffering from the disease, decreasing the dose of other medicationsrequired to treat the disease, enhancing effect of another medicationsuch as via targeting, delaying the progression of the disease, and/orprolonging survival. In the case of cancer or tumor, an effective amountof the drug may have the effect in reducing the number of cancer cells;reducing the tumor size; inhibiting (i.e., slow to some extent ordesirably stop) cancer cell infiltration into peripheral organs; inhibit(i.e., slow to some extent and desirably stop) tumor metastasis;inhibiting to some extent tumor growth; and/or relieving to some extentone or more of the symptoms associated with the disorder. An effectiveamount can be administered in one or more administrations. For purposesof this invention, an effective amount of drug, compound, orpharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective amount of a drug,compound, or pharmaceutical composition may or may not be achieved inconjunction with another drug, compound, or pharmaceutical composition.Thus, an “effective amount” may be considered in the context ofadministering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction withone or more other agents, a desirable result may be or is achieved.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In some embodiments, an FcR is a native human FcR. Insome embodiments, an FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof those receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see, e.g., Daeron,Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example,in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term “Fc receptor”or “FcR” also includes the neonatal receptor, FcRn, which is responsiblefor the transfer of maternal IgGs to the fetus (Guyer et al., J.Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) andregulation of homeostasis of immunoglobulins. Methods of measuringbinding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today18(12):592-598 (1997); Ghetie et al., Nature Biotechnology,15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216(2004); WO 2004/92219 (Hinton et al.). Binding to human FcRn in vivo andserum half life of human FcRn high affinity binding polypeptides can beassayed, e.g., in transgenic mice or transfected human cell linesexpressing human FcRn, or in primates to which the polypeptides with avariant Fc region are administered. WO 2000/42072 (Presta) describesantibody variants with improved or diminished binding to FcRs. See also,e.g., Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include C1q binding;CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cellsurface receptors (e.g. B cell receptor; BCR), etc. Such effectorfunctions generally require the Fc region to be combined with a bindingdomain (e.g., an antibody variable domain) and can be assessed usingvarious assays as disclosed, for example, in definitions herein.

“Human effector cells” refer to leukocytes that express one or more FcRsand perform effector functions. In certain embodiments, the cellsexpress at least FcγRIII and perform ADCC effector function(s). Examplesof human leukocytes which mediate ADCC include peripheral bloodmononuclear cells (PBMC), natural killer (NK) cells, monocytes,cytotoxic T cells, and neutrophils. The effector cells may be isolatedfrom a native source, e.g., from blood.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs: three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothiaand Lesk, J. Mol. Biol. 196:901-917 (1987));(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97(L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991));(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55(L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum etal. J. Mol. Biol. 262: 732-745 (1996)); and(d) combinations of (a), (b), and/or (c), including HVR amino acidresidues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1),26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

“Promoting cell growth or proliferation” means increasing a cell'sgrowth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 100%.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-OX40 antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain. A“native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during, or after administration of the other treatment modalityto the individual. For example, an anti-angiogenesis agent may beadministered in conjunction with an OX40 binding agonist. Ananti-angiogenesis agent and an OX40 binding agonist may be administeredin conjunction with another a chemotherapeutic agent.

The term “tumor” refers to all neoplastic cell growth and proliferation,whether malignant or benign, and all pre-cancerous and cancerous cellsand tissues. The terms “cancer,” “cancerous,” “cell proliferativedisorder,” “proliferative disorder” and “tumor” are not mutuallyexclusive as referred to herein.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, preferably one or more amino acid substitution(s).Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g. from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide, and most preferably at least about90% homology therewith, more preferably at least about 95% homologytherewith.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al. In one embodiment, the VH subgroup III consensus frameworkamino acid sequence comprises at least a portion or all of each of thefollowing sequences: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ IDNO:222)-H1-WVRQAPGKGLEWV (SEQ IDNO:223)-H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO:224)-H3-WGQGTLVTVSS(SEQ ID NO:225).

A “VL subgroup I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al. In one embodiment, the VH subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences:

DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 226)-L1-WYQQKPGKAPKLLIY (SEQ ID NO: 227)-L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 228)-L3-FGQGTKVEIK (SEQ ID NO: 229).

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153,Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeuticagents; growth inhibitory agents; enzymes and fragments thereof such asnucleolytic enzymes; and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof. Exemplary cytotoxicagents can be selected from anti-microtubule agents, platinumcoordination complexes, alkylating agents, antibiotic agents,topoisomerase II inhibitors, antimetabolites, topoisomerase Iinhibitors, hormones and hormonal analogues, signal transduction pathwayinhibitors, non-receptor tyrosine kinase angiogenesis inhibitors,immunotherapeutic agents, proapoptotic agents, inhibitors of LDH-A;inhibitors of fatty acid biosynthesis; cell cycle signalling inhibitors;HDAC inhibitors, proteasome inhibitors; and inhibitors of cancermetabolism.

In one embodiment the cytotoxic agent is selected from anti-microtubuleagents, platinum coordination complexes, alkylating agents, antibioticagents, topoisomerase II inhibitors, antimetabolites, topoisomerase Iinhibitors, hormones and hormonal analogues, signal transduction pathwayinhibitors, non-receptor tyrosine kinase angiogenesis inhibitors,immunotherapeutic agents, proapoptotic agents, inhibitors of LDH-A,inhibitors of fatty acid biosynthesis, cell cycle signalling inhibitors,HDAC inhibitors, proteasome inhibitors, and inhibitors of cancermetabolism. In one embodiment the cytotoxic agent is a taxane. In oneembodiment the taxane is paclitaxel or docetaxel. In one embodiment thecytotoxic agent is a platinum agent. In one embodiment the cytotoxicagent is an antagonist of EGFR. In one embodiment the antagonist of EGFRis N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (e.g.,erlotinib). In one embodiment the cytotoxic agent is a RAF inhibitor. Inone embodiment, the RAF inhibitor is a BRAF and/or CRAF inhibitor. Inone embodiment the RAF inhibitor is vemurafenib. In one embodiment thecytotoxic agent is a PI3K inhibitor.

“Chemotherapeutic agent” includes chemical compounds useful in thetreatment of cancer. Examples of chemotherapeutic agents includeerlotinib (TARCEVA®, Genentech/OSI Pharm.), bortezomib (VELCADE®,Millennium Pharm.), disulfiram, epigallocatechin gallate,salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol,lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca),sunitib (SUTENT®, Pfizer/Sugen), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), finasunate (VATALANIB®, Novartis),oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin,Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (TYKERB®, GSK572016,Glaxo Smith Kline), Lonafamib (SCH 66336), sorafenib (NEXAVAR®, BayerLabs), gefitinib (IRESSA®, AstraZeneca), AG1478, alkylating agents suchas thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (includingtopotecan and irinotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);adrenocorticosteroids (including prednisone and prednisolone);cyproterone acetate; 5α-reductases including finasteride anddutasteride); vorinostat, romidepsin, panobinostat, valproic acid,mocetinostat dolastatin; aldesleukin, talc duocarmycin (including thesynthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; asarcodictyin; spongistatin; nitrogen mustards such as chlorambucil,chlomaphazine, chlorophosphamide, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; antibiotics such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin yll andcalicheamicin ω1I (Angew Chem. Intl. Ed. Engl. 1994 33:183-186);dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®(doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL(paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® (docetaxel, doxetaxel; Sanofi-Aventis); chloranmbucil; GEMZAR®(gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; etoposide(VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE®(vinorelbine); novantrone; teniposide; edatrexate; daunomycin;aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; topoisomeraseinhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such asretinoic acid; and pharmaceutically acceptable salts, acids andderivatives of any of the above.

Chemotherapeutic agent also includes (i) anti-hormonal agents that actto regulate or inhibit hormone action on tumors such as anti-estrogensand selective estrogen receptor modulators (SERMs), including, forexample, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene,droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and FARESTON® (toremifine citrate); (ii)aromatase inhibitors that inhibit the enzyme aromatase, which regulatesestrogen production in the adrenal glands, such as, for example,4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate),AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR®(vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole;AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide and goserelin; buserelin, tripterelin,medroxyprogesterone acetate, diethylstilbestrol, premarin,fluoxymesterone, all transretionic acid, fenretinide, as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors; (v) lipid kinase inhibitors; (vi) antisenseoligonucleotides, particularly those which inhibit expression of genesin signaling pathways implicated in aberrant cell proliferation, suchas, for example, PKC-alpha, Ralf and H-Ras; (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN®, rIL-2; a topoisomerase 1 inhibitorsuch as LURTOTECAN®; ABARELIX® rmRH; and (ix) pharmaceuticallyacceptable salts, acids and derivatives of any of the above.

Chemotherapeutic agent also includes antibodies such as alemtuzumab(Campath), cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®,Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec), pertuzumab(OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech),tositumomab (Bexxar, Corixia), and the antibody drug conjugate,gemtuzumab ozogamicin (MYLOTARG®, Wyeth). Additional humanizedmonoclonal antibodies with therapeutic potential as agents incombination with the compounds of the invention include: apolizumab,aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumabmertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab,daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab,fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab,labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab,motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab,ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab,pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab,reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab,siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab,tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin,tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, andthe anti-interleukin-12 (ABT-874/J695, Wyeth Research and AbbottLaboratories) which is a recombinant exclusively human-sequence,full-length IgG1λ antibody genetically modified to recognizeinterleukin-12 p40 protein.

Chemotherapeutic agent also includes “EGFR inhibitors,” which refers tocompounds that bind to or otherwise interact directly with EGFR andprevent or reduce its signaling activity, and is alternatively referredto as an “EGFR antagonist.” Examples of such agents include antibodiesand small molecules that bind to EGFR. Examples of antibodies which bindto EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507),MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat. No.4,943,533, Mendelsohn et al.) and variants thereof, such as chimerized225 (C225 or Cetuximab; ERBUTIX®) and reshaped human 225 (H225) (see, WO96/40210, Imclone Systems Inc.); IMC-11F8, a fully human, EGFR-targetedantibody (Imclone); antibodies that bind type II mutant EGFR (U.S. Pat.No. 5,212,290); humanized and chimeric antibodies that bind EGFR asdescribed in U.S. Pat. No. 5,891,996; and human antibodies that bindEGFR, such as ABX-EGF or Panitumumab (see WO98/50433, Abgenix/Amgen);EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996));EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR thatcompetes with both EGF and TGF-alpha for EGFR binding (EMD/Merck); humanEGFR antibody, HuMax-EGFR (GenMab); fully human antibodies known asE1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6. 3 and E7.6. 3 and described inU.S. Pat. No. 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanizedmAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). Theanti-EGFR antibody may be conjugated with a cytotoxic agent, thusgenerating an immunoconjugate (see, e.g., EP659,439A2, Merck PatentGmbH). EGFR antagonists include small molecules such as compoundsdescribed in U.S. Pat. Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307,5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726,6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459,6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, aswell as the following PCT publications: WO98/14451, WO98/50038,WO99/09016, and WO99/24037. Particular small molecule EGFR antagonistsinclude OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech/OSIPharmaceuticals); PD 183805 (CI 1033, 2-propenamide,N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-,dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®)4-(3′-Chloro-4′-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline,AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline,Zeneca); BIBX-1382(N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine,Boehringer Ingelheim); PKI-166((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol);(R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine);CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide);EKB-569(N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide)(Wyeth); AG1478 (Pfizer); AG1571 (SU 5271; Pfizer); dual EGFR/HER2tyrosine kinase inhibitors such as lapatinib (TYKERB®, GSK572016 orN-[3-chloro-4-[(3fluorophenyl)methoxy]phenyl]-6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine).

Chemotherapeutic agents also include “tyrosine kinase inhibitors”including the EGFR-targeted drugs noted in the preceding paragraph;small molecule HER2 tyrosine kinase inhibitor such as TAK165 availablefrom Takeda; CP-724,714, an oral selective inhibitor of the ErbB2receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such asEKB-569 (available from Wyeth) which preferentially binds EGFR butinhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016;available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinaseinhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such ascanertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as antisenseagent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1signaling; non-HER targeted TK inhibitors; multi-targeted tyrosinekinase inhibitors such as sunitinib (SUTENT®, available from Pfizer);VEGF receptor tyrosine kinase inhibitors such as vatalanib(PTK787/ZK222584, available from Novartis/Schering AG); MAPKextracellular regulated kinase I inhibitor CI-1040 (available fromPharmacia); quinazolines, such as PD 153035,4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines,such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines,4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines; curcumin (diferuloylmethane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines containingnitrothiophene moieties; PD-0183805 (Warner-Lamber); antisense molecules(e.g. those that bind to HER-encoding nucleic acid); quinoxalines (U.S.Pat. No. 5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474(Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors suchas CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); imatinibmesylate (GLEEVEC®); PKI 166 (Novartis); GW2016 (Glaxo SmithKline);CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474(AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone),rapamycin (sirolimus, RAPAMUNE®); or as described in any of thefollowing patent publications: U.S. Pat. No. 5,804,396; WO 1999/09016(American Cyanamid); WO 1998/43960 (American Cyanamid); WO 1997/38983(Warner Lambert); WO 1999/06378 (Warner Lambert); WO 1999/06396 (WarnerLambert); WO 1996/30347 (Pfizer, Inc); WO 1996/33978 (Zeneca); WO1996/3397 (Zeneca) and WO 1996/33980 (Zeneca).

Chemotherapeutic agents also include dexamethasone, interferons,colchicine, metoprine, cyclosporine, amphotericin, metronidazole,alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide,asparaginase, BCG live, bevacuzimab, bexarotene, cladribine,clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa,elotinib, filgrastim, histrelin acetate, ibritumomab, interferonalfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna,methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin,palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim,pemetrexed disodium, plicamycin, porfimer sodium, quinacrine,rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene,tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, andpharmaceutically acceptable salts thereof.

Chemotherapeutic agents also include hydrocortisone, hydrocortisoneacetate, cortisone acetate, tixocortol pivalate, triamcinoloneacetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide,desonide, fluocinonide, fluocinolone acetonide, betamethasone,betamethasone sodium phosphate, dexamethasone, dexamethasone sodiumphosphate, fluocortolone, hydrocortisone-17-butyrate,hydrocortisone-17-valerate, aclometasone dipropionate, betamethasonevalerate, betamethasone dipropionate, prednicarbate,clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolonecaproate, fluocortolone pivalate and fluprednidene acetate; immuneselective anti-inflammatory peptides (ImSAIDs) such asphenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG)(IMULAN BioTherapeutics, LLC); anti-rheumatic drugs such asazathioprine, ciclosporin (cyclosporine A), D-penicillamine, gold salts,hydroxychloroquine, leflunomideminocycline, sulfasalazine, tumornecrosis factor alpha (TNFa) blockers such as etanercept (Enbrel),infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia),golimumab (Simponi), Interleukin 1 (IL-1) blockers such as anakinra(Kineret), T cell costimulation blockers such as abatacept (Orencia),Interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERA®);Interleukin 13 (IL-13) blockers such as lebrikizumab; Interferon alpha(IFN) blockers such as Rontalizumab; Beta 7 integrin blockers such asrhuMAb Beta7; IgE pathway blockers such as Anti-M1 prime; Secretedhomotrimeric LTa3 and membrane bound heterotrimer LTal/32 blockers suchas Anti-lymphotoxin alpha (LTa); radioactive isotopes (e.g., At211,I1131, I1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 andradioactive isotopes of Lu); miscellaneous investigational agents suchas thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832); polyphenols such asquercetin, resveratrol, piceatannol, epigallocatechine gallate,theaflavins, flavanols, procyanidins, betulinic acid and derivativesthereof; autophagy inhibitors such as chloroquine;delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone;lapachol; colchicines; betulinic acid; acetylcamptothecin, scopolectin,and 9-aminocamptothecin); podophyllotoxin; tegafur (UFTORAL®);bexarotene (TARGRETIN®); bisphosphonates such as clodronate (forexample, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095,zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®),pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine; perifosine, COX-2 inhibitor (e.g. celecoxib oretoricoxib), proteosome inhibitor (e.g. PS341); CCI-779; tipifarnib(R11577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium(GENASENSE®); pixantrone; farnesyltransferase inhibitors such aslonafarnib (SCH 6636, SARASAR™); and pharmaceutically acceptable salts,acids or derivatives of any of the above; as well as combinations of twoor more of the above such as CHOP, an abbreviation for a combinedtherapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone;and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovorin.

Chemotherapeutic agents also include non-steroidal anti-inflammatorydrugswith analgesic, antipyretic and anti-inflammatory effects. NSAIDsinclude non-selective inhibitors of the enzyme cyclooxygenase. Specificexamples of NSAIDs include aspirin, propionic acid derivatives such asibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen,acetic acid derivatives such as indomethacin, sulindac, etodolac,diclofenac, enolic acid derivatives such as piroxicam, meloxicam,tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivativessuch as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamicacid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib,parecoxib, rofecoxib, rofecoxib, and valdecoxib. NSAIDs can be indicatedfor the symptomatic relief of conditions such as rheumatoid arthritis,osteoarthritis, inflammatory arthropathies, ankylosing spondylitis,psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhoea,metastatic bone pain, headache and migraine, postoperative pain,mild-to-moderate pain due to inflammation and tissue injury, pyrexia,ileus, and renal colic.

The term “cytokine” is a generic term for proteins released by one cellpopulation that act on another cell as intercellular mediators. Examplesof such cytokines are lymphokines, monokines; interleukins (ILs) such asIL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11,IL-12, IL-15; a tumor necrosis factor such as TNF-α or TNF-3; and otherpolypeptide factors including LIF and kit ligand (KL) and gammainterferon. As used herein, the term cytokine includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native-sequence cytokines, including syntheticallyproduced small-molecule entities and pharmaceutically acceptablederivatives and salts thereof.

An “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to asmall molecular weight substance, a polynucleotide, a polypeptide, anisolated protein, a recombinant protein, an antibody, or conjugates orfusion proteins thereof, that inhibits angiogenesis, vasculogenesis, orundesirable vascular permeability, either directly or indirectly. Itshould be understood that the anti-angiogenesis agent includes thoseagents that bind and block the angiogenic activity of the angiogenicfactor or its receptor. For example, an anti-angiogenesis agent is anantibody or other antagonist to an angiogenic agent as definedthroughout the specification or known in the art, e.g., but are notlimited to, antibodies to VEGF-A or to the VEGF-A receptor (e.g., KDRreceptor or Flt-1 receptor), VEGF-trap, anti-PDGFR inhibitors such asGleevec™ (Imatinib Mesylate). Anti-angiogenesis agents also includenative angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See,e.g., Klagsbrun and D'Amore, Annu. Rev. Physiol., 53:217-39 (1991);Streit and Detmar, Oncogene, 22:3172-3179 (2003) (e.g., Table 3 listinganti-angiogenic therapy in malignant melanoma); Ferrara & Alitalo,Nature Medicine 5:1359-1364 (1999); Tonini et al., Oncogene,22:6549-6556 (2003) (e.g., Table 2 listing known antiangiogenicfactors); and Sato. Int. J. Clin. Oncol., 8:200-206 (2003) (e.g., Table1 lists anti-angiogenic agents used in clinical trials).

The term “VEGF” or “VEGF-A” is used to refer to the 165-amino acid humanvascular endothelial cell growth factor and related 121-, 145-, 189-,and 206-amino acid human vascular endothelial cell growth factors, asdescribed by, e.g., Leung et al. Science, 246:1306 (1989), and Houck etal. Mol. Endocrin., 5:1806 (1991), together with the naturally occurringallelic and processed forms thereof. VEGF-A is part of a gene familyincluding VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and P1GF. VEGF-Aprimarily binds to two high affinity receptor tyrosine kinases, VEGFR-1(Flt-1) and VEGFR-2 (Flk-1/KDR), the latter being the major transmitterof vascular endothelial cell mitogenic signals of VEGF-A. Additionally,neuropilin-1 has been identified as a receptor for heparin-bindingVEGF-A isoforms, and may play a role in vascular development. The term“VEGF” or “VEGF-A” also refers to VEGFs from non-human species such asmouse, rat, or primate. Sometimes the VEGF from a specific species isindicated by terms such as hVEGF for human VEGF or mVEGF for murineVEGF. Typically, VEGF refers to human VEGF. The term “VEGF” is also usedto refer to truncated forms or fragments of the polypeptide comprisingamino acids 8 to 109 or 1 to 109 of the 165-amino acid human vascularendothelial cell growth factor. Reference to any such forms of VEGF maybe identified in the application, e.g., by “VEGF (8-109),” “VEGF(1-109)” or “VEGF165.” The amino acid positions for a “truncated” nativeVEGF are numbered as indicated in the native VEGF sequence. For example,amino acid position 17 (methionine) in truncated native VEGF is alsoposition 17 (methionine) in native VEGF. The truncated native VEGF hasbinding affinity for the KDR and Flt-1 receptors comparable to nativeVEGF.

A “chimeric VEGF receptor protein” is a VEGF receptor molecule havingamino acid sequences derived from at least two different proteins, atleast one of which is a VEGF receptor protein. In certain embodiments,the chimeric VEGF receptor protein is capable of binding to andinhibiting the biological activity of VEGF.

A “VEGF antagonist” or “VEGF-specific antagonist” refers to a moleculecapable of binding to VEGF, reducing VEGF expression levels, orneutralizing, blocking, inhibiting, abrogating, reducing, or interferingwith VEGF biological activities, including, but not limited to, VEGFbinding to one or more VEGF receptors, VEGF signaling, and VEGF mediatedangiogenesis and endothelial cell survival or proliferation. Forexample, a molecule capable of neutralizing, blocking, inhibiting,abrogating, reducing, or interfering with VEGF biological activities canexert its effects by binding to one or more VEGF receptor (VEGFR) (e.g.,VEGFR1. VEGFR2. VEGFR3, mermbrane-bound VEGF receptor (mbVEGFR), orsoluble VEGF receptor (sVEGFR)). Included as VEGF-specific antagonistsuseful in the methods of the invention are polypeptides thatspecifically bind to VEGF, anti-VEGF antibodies and antigen-bindingfragments thereof, receptor molecules and derivatives which bindspecifically to VEGF thereby sequestering its binding to one or morereceptors, fusions proteins (e.g., VEGF-Trap (Regeneron)), andVEGF₁₂₁-gelonin (Peregrine). VEGF-specific antagonists also includeantagonist variants of VEGF polypeptides, antisense nucleobase oligomerscomplementary to at least a fragment of a nucleic acid molecule encodinga VEGF polypeptide; small RNAs complementary to at least a fragment of anucleic acid molecule encoding a VEGF polypeptide; ribozymes that targetVEGF; peptibodies to VEGF; and VEGF aptamers. VEGF antagonists alsoinclude polypeptides that bind to VEGFR, anti-VEGFR antibodies, andantigen-binding fragments thereof, and derivatives which bind to VEGFRthereby blocking, inhibiting, abrogating, reducing, or interfering withVEGF biological activities (e.g., VEGF signaling), or fusions proteins.VEGF-specific antagonists also include nonpeptide small molecules thatbind to VEGF or VEGFR and are capable of blocking, inhibiting,abrogating, reducing, or interfering with VEGF biological activities.Thus, the term “VEGF activities” specifically includes VEGF mediatedbiological activities of VEGF. In certain embodiments, the VEGFantagonist reduces or inhibits, by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or more, the expression level or biological activityof VEGF. In some embodiments, the VEGF inhibited by the VEGF-specificantagonist is VEGF (8-109). VEGF (1-109), or VEGF₁₆₅.

As used herein VEGF antagonists can include, but are not limited to,anti-VEGFR2 antibodies and related molecules (e.g., ramucirumnab,tanibirumab, aflibercept), anti-VEGFR1 antibodies and related molecules(e.g., icrucumab, aflibercept (VEGF Trap-Eye; EYLEA®), andziv-aflibercept (VEGF Trap; ZALTRAP®)), bispecific VEGF antibodies(e.g., MP-0250, vanucizumab (VEGF-ANG2), and bispecific antibodiesdisclosed in US 2001/0236388), bispecific antibodies includingcombinations of two of anti-VEGF, anti-VEGFR1, and anti-VEGFR2 arms,anti-VEGFA antibodies (e.g., bevacizumab, sevacizumab), anti-VEGFBantibodies, anti-VEGFC antibodies (e.g., VGX-100), anti-VEGFDantibodies, and nonpeptide small molecule VEGF antagonists (e.g.,pazopanib, axitinib, vandetanib, stivarga, cabozantinib, lenvatinib,nintedanib, orantinib, telatinib, dovitinig, cediranib, motesanib,sulfatinib, apatinib, foretinib, famitinib, and tivozanib).

An “anti-VEGF antibody” is an antibody that binds to VEGF withsufficient affinity and specificity. In certain embodiments, theantibody will have a sufficiently high binding affinity for VEGF, forexample, the antibody may bind hVEGF with a K_(d) value of between 100nM-1 pM. Antibody affinities may be determined, e.g., by a surfaceplasinon resonance based assay (such as the BIAcore assay as describedin PCT Application Publication No. WO2005/012359); enzyme-linkedimmunoabsorbent assay (ELISA); and competition assays (e.g. RIA's). Incertain embodiments, the anti-VEGF antibody can be used as a therapeuticagent in targeting and interfering with diseases or conditions whereinthe V^(E) F activity is involved. Also, the antibody may be subjected toother biological activity assays, e.g., in order to evaluate itseffectiveness as a therapeutic. Such assays are known in the art anddepend on the target antigen and intended use for the antibody. Examplesinclude the HUVEC inhibition assay; tumor cell growth inhibition assays(as described in WO 89/06692, for example); antibody-dependent cellularcytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays(U.S. Pat. No. 5,500,362); and agonistic activity or hematopoiesisassays (see WO 95/27062). Anr anti-VEGF antibody will usually not bindto other VEGF homologues such as VEGF-B or VEGF-C, nor other growthfactors such as PIGF, PDGF, or bFGF. In one embodiment, anti-VEGFantibody is a monoclonal antibody that binds to the same epitope as themonoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB10709. In another embodiment, the anti-VEGF antibody is a recombinanthumanized anti-VEGF monoclonal antibody generated according to Presta etal. (1997) Cancer Res. 57:4593-4599, including but not limited to theantibody known as bevacizumab (BV; AVASTIN®).

The anti-VEGF antibody “Bevacizumab (BV),” also known as “rhuMAb VEGF”or “AVASTINY,” is a recombinant humanized anti-VEGF monoclonal antibodygenerated according to Presta et al. (1997) Cancer Res. 57:4593-4599. Itcomprises mutated human igG1 framework regions anrid antigen-bindingcomplementarity-determining regions from the murine anti-hVEGFmonoclonal antibody A.4.6.1 that blocks binding of human VEGF to itsreceptors. Approximately 93% of the amino acid sequence of bevacizumab,including most of the framework regions, is derived from human IgG1, andabout 7% of the sequence is derived from the iurine antibody A4.6.1.Bevacizumab has a molecular mass of about 149,000 daltons and isglycosylated. Bevacizumab and other humanized anti-VEGF antibodies arefurther described in U.S. Pat. No. 6,884,879 issued Feb. 26, 2005, theentire disclosure of which is expressly incorporated herein byreference. Additional preferred antibodies include the G6 or B20 seriesantibodies (e.g., G6-31, B20-4.1), as described in PCT ApplicationPublication No. WO 2005/012359. For additional preferred antibodies seeU.S. Pat. Nos. 7,060,269, 6,582,959, 6,703,020; 6,054,297; WO98/45332;WO 96/30046; WO94/10202; EP 0666868B1; U.S. Patent ApplicationPublication Nos. 2006009360, 20050186208, 20030206899, 20030190317,20030203409, and 20050112126; and Popkov et al., Journal ofImmunological Methods 288:149-164 (2004). Other preferred antibodiesinclude those that bind to a functional epitope on human VEGF comprisingof residues F17, M18, D19, Y21, Y25, Q89, 191, K101, E103, and C104 or,alternatively, comprising residues F17, Y21, Q22, Y25, D63, 183, andQ89.

The “epitope A4.6.1” refers to the epitope recognized by the anti-VEGFantibody bevacizumab (AVASTIN®) (see Muller Y et al., Structure 15 Sep.1998, 6:1153-1167). In certain embodiments of the invention, theanti-VEGF antibodies include, but are not limited to, a monoclonalantibody that binds to the same epitope as the monoclonal anti-VEGFantibody A4.6.1 produced by hybridoma ATCC HB 10709; a recombinanthumanized anti-VEGF monoclonal antibody generated according to Presta etal. (1997) Cancer Res. 57:4593-4599.

By “standard of care” herein is intended the anti-tumor agent or agentsthat are routinely used to treat a particular form of cancer. Forexample, for platinum-resistant ovarian cancer, a standard of care istopotecan or liposomal doxorubicin.

By “platinum-based chemotherapeutic agent” or “platin” is meant anantineoplastic drug that is a coordination complex of platinum. Examplesof platinum-based chemotherapeutic agents include carboplatin,cisplatin, and oxaliplatinum.

By “platinum-based chemotherapy” is meant therapy with one or moreplatinum-based chemotherapeutic agent, optionally in combination withone or more other chemotherapeutic agents.

By “chemotherapy-resistant” cancer is meant cancer in a patient that hasprogressed while the patient is receiving a chemotherapy regimen (i.e.,the patient is “chemotherapy refractory”), or the patient has progressedwithin 12 months (for instance, within 6 months) after completing achemotherapy regimen.

By “platinum-resistant” cancer is meant cancer in a patient that hasprogressed while receiving platinum-based chemotherapy (i.e., thepatient is “platinum refractory”), or the patient has progressed within12 months (for instance, within 6 months) after completing aplatinum-based chemotherapy regimen.

By “radiation therapy” is meant the use of directed gamma rays or betarays to induce sufficient damage to a cell so as to limit its ability tofimction normally or to destroy the cell altogether. It will beappreciated that there will be many ways known in the art to determinethe dosage and duration of treatment. Typical treatments are given as aone-time administration and typical dosages range from 10 to 200 units(Grays) per day.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a molecule”optionally includes a combination of two or more such molecules, and thelike.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

II. Anti-Angiogenesis Agents

Provided herein are methods treating or delaying progression of cancerin an individual comprising administering to the individual an effectiveamount of an anti-angiogenesis agent and an OX40 binding agonist.

As described supra, an anti-angiogenesis agent may include a compoundsuch as a small molecular weight substance, a polynucleotide, apolypeptide, an isolated protein, a recombinant protein, an antibody, orconjugates or fusion proteins thereof. In some embodiments, theanti-angiogenesis agent is an anti-VEGFR2 antibody; an anti-VEGFR1antibody; a VEGF-trap; a bispecific VEGF antibody; a bispecific antibodycomprising a combination of two arms selected from an anti-VEGF arm, ananti-VEGFR1 arm, and an anti-VEGFR2 arm; an anti-VEGF-A antibody (e.g.,an anti-KDR receptor or anti-Flt-1 receptor antibody); an anti-VEGFBantibody; an anti-VEGFC antibody; an anti-VEGFD antibody; a nonpeptidesmall molecule VEGF antagonist; an anti-PDGFR inhibitor; or a nativeangiogenesis inhibitor. In certain embodiments, the anti-angiogenesisagent is ramucirumab, tanibirumab, aflibercept (e.g., VEGF Trap-Eye;EYLEA®), icrucumab, ziv-aflibercept (e.g., VEGF Trap; ZALTRAP®),MP-0250, vanucizumab, sevacizumab, VGX-100, pazopanib, axitinib,vandetanib, stivarga, cabozantinib, lenvatinib, nintedanib, orantinib,telatinib, dovitinig, cediranib, motesanib, sulfatinib, apatinib,foretinib, famitinib, imatinib (e.g., Imatinib Mesylate; Gleevec™), andtivozanib.

In some embodiments, the anti-angiogenesis agent is an anti-angiogenesisantibody. Descriptions of antibodies and methods for generatingantibodies are further provided infra. In some embodiments, theanti-angiogenesis antibody is a monoclonal antibody. In someembodiments, the anti-angiogenesis antibody is a human or humanizedantibody (described in more detail below).

In some embodiments, the anti-angiogenesis agent is a VEGF antagonist.For example, VEGF antagonists of the present disclosure may includewithout limitation polypeptides that specifically bind to VEGF,anti-VEGF antibodies and antigen-binding fragments thereof; receptormolecules and derivatives which bind specifically to VEGF, therebysequestering its binding to one or more receptors; fusion proteins(e.g., VEGF-Trap (Regeneron)), VEGF₁₂₁-gelonin (Peregrine), antagonistvariants of VEGF polypeptides, antisense nucleobase oligomerscomplementary to at least a fragment of a nucleic acid molecule encodinga VEGF polypeptide; small RNAs complementary to at least a fragment of anucleic acid molecule encoding a VEGF polypeptide (e.g., an RNAi, siRNA,shRNA, or miRNA); ribozymes that target VEGF; peptibodies to VEGF; VEGFaptamers; polypeptides that bind to VEGFR; anti-VEGFR antibodies andantigen-binding fragments thereof; derivatives which bind to VEGFRthereby blocking, inhibiting, abrogating, reducing, or interfering withVEGF biological activities (e.g., VEGF signaling); fusion proteins; andnonpeptide small molecules that bind to VEGF or VEGFR and are capable ofblocking, inhibiting, abrogating, reducing, or interfering with VEGFbiological activities.

In certain embodiments, the VEGF antagonist reduces or inhibits, by atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, theexpression level or biological activity of VEGF. For example, in someembodiments, the VEGF antagonist may reduce or inhibit the expressionlevel or biological activity of VEGF by at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%. In some embodiments, the VEGF inhibited by the VEGF-specificantagonist is VEGF (8-109). VEGF (1-109), or VEGFs₅.

Certain aspects of the methods, uses, and kits of the present disclosureare based, at least in part, on the surprising discovery that anti-VEGFtreatment can improve the functional phenotype of tumoral dendriticcells (e.g., by leading to increased expression of MHC Class II and/orOX40L). Without wishing to be bound to theory, this property, interalia, may make combination therapies including an anti-angiogenesisagent and an OX40 binding agonist particularly advantageous for thetreatment of cancer, e.g., by resulting in enhanced anti-tumor responsessuch as anti-tumoral T cell responses.

Therefore, in some embodiments, the VEGF antagonist increases MHC classII expression on intratumoral dendritic cells, e.g., as compared to MHCclass II expression on dendritic cells from a tumor treated with acontrol antibody (e.g., an isotype control). MHC class II is known as afamily of related molecules (typically heterodimers containing alpha andbeta chains) that present antigen to T cells. As used herein, MHC classII expression may refer to expression of any MHC class II molecule orchain, including without limitation a polypeptide encoded by the humangenes HLA-DM alpha (e.g., NCBI Gene ID No. 3108), HLA-DM beta (e.g.,NCBI Gene ID No. 3109), HLA-DO alpha (e.g., NCBI Gene ID No. 3111),HLA-DO beta (e.g., NCBI Gene ID No. 3112), HLA-DP alpha 1 (e.g., NCBIGene ID No. 3113), HLA-DP beta 1 (e.g., NCBI Gene ID No. 3115), HLA-DQalpha 1 (e.g., NCBI Gene ID No. 3117), HLA-DQ alpha 2 (e.g., NCBI GeneID No. 3118), HLA-DQ beta 1 (e.g., NCBI Gene ID No. 3119), HLA-DQ beta 2(e.g., NCBI Gene ID No. 3120), HLA-DR alpha (e.g., NCBI Gene ID No.3122), HLA-DR beta 1 (e.g., NCBI Gene ID No. 3123), HLA-DR beta 3 (e.g.,NCBI Gene ID No. 3125), HLA-DR beta 4 (e.g., NCBI Gene ID No. 3126), orHLA-DR beta 5 (e.g., NCBI Gene ID No. 3127). It will be appreciated byone of skill in the art that MHC genes are highly variable acrosspopulations, and thus the specific genes and sequences listed are merelyexemplary and in no way intended to be limiting.

In some embodiments, the VEGF antagonist increases OX40L expression onintratumoral dendritic cells, e.g., as compared to OX40L expression ondendritic cells from a tumor treated with a control antibody (e.g., anisotype control). OX40L (also known as tumor necrosis factor ligandsuperfamily member 4 or CD252) is known as the binding partner or ligandof OX40. Examples of OX40L polypeptides including without limitationpolypeptides having the amino acid sequence represented by UniProtAccession No. P43488 and/or a polypeptide encoded by gene TNFSF4 (e.g.,NCBI Gene ID No. 7292).

Methods for measuring MHC class II or OX40L expression are known in theart and may include without limitation FACS, Western blot, ELISA,immunoprecipitation, immunohistochemistry, immunofluorescence,radioimmunoassay, dot blotting, immunodetection methods, HPLC, surfaceplasmon resonance, optical spectroscopy, mass spectrometery, HPLC, qPCR,RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE,MassARRAY technique, and FISH, and combinations thereof.

In some embodiments, the dendritic cells are myeloid dendritic cells. Inother embodiments, the dendritic cells are non-myeloid dendritic cells(e.g., lymphoid or plasmacytoid dendritic cells). The cell-surfaceantigens expressed by dendritic cells, and those that distinguishmyeloid and non-myeloid dendritic cells, are known in the art. Forexample, dendritic cells may be identified by expression of CD45, CD1 c,and MHC class II. They may be distinguished from other cell types (e.g.,macrophages, neutrophils, and granulocytic myeloid cells) by their lackof significant F4/80 and Gr1expression. In some embodiments, myeloiddendritic cells are dendritic cells that express CD11b, and non-myeloiddendritic cells are dendritic cells that lack significant CD11bexpression. For further descriptions of myeloid and non-myeloiddendritic cells, see, e.g., Steinman, R. M. and Inaba, K. (1999) J.Leukoc. Biol. 66:205-8.

VEGF Receptor Molecules

In some embodiments, the anti-angiogenesis agent is a VEGF antagonist.In some embodiments, the VEGF antagonist comprises a soluble VEGFreceptor or a soluble VEGF receptor fragment that specifically binds toVEGF. The two best characterized VEGF receptors are VEGFR1 (also knownas Flt-1) and VEGFR2 (also known as KDR and FLK-1 for the murinehomolog). The specificity of each receptor for each VEGF family membervaries but VEGF-A binds to both Flt-1 and KDR. Both Flt-I and KDR belongto the family of receptor tyrosine kinases (RTKs). The RTKs comprise alarge family of transmembrane receptors with diverse biologicalactivities. At least nineteen (19) distinct RTK subfamilies have beenidentified. The receptor tyrosine kinase (RTK) family includes receptorsthat are crucial for the growth and differentiation of a variety of celltypes (Yarden and Ullrich (1988) Ann. Rev. Biochem. 57:433-478; Ullrichand Schlessinger (1990) Cell 61:243-254). The intrinsic function of RTKsis activated upon ligand binding, which results in phosphorylation ofthe receptor and multiple cellular substrates, and subsequently in avariety of cellular responses (Ullrich & Schlessinger (1990) Cell61:203-212). Thus, receptor tyrosine kinase mediated signal transductionis initiated by extracellular interaction with a specific growth factor(ligand), typically followed by receptor dimerization, stimulation ofthe intrinsic protein tyrosine kinase activity and receptortrans-phosphorylation. Binding sites are thereby created forintracellular signal transduction molecules and lead to the formation ofcomplexes with a spectrum of cytoplasmic signaling molecules thatfacilitate the appropriate cellular response. (e.g., cell division,differentiation, metabolic effects, changes in the extracellularmicroenvironment) see, Schlessinger and Ullrich (1992) Neuron 9:1-20.Structurally, both Flt-1 and KDR have seven immunoglobulin-like domainsin the extracellular domain, a single transmembrane region, and aconsensus tyrosine kinase sequence which is interrupted by akinase-insert domain. Matthews et al. (1991) PNAS USA 88:9026-9030;Terman et al. (1991) Oncogene 6:1677-1683. The extracellular domain isinvolved in the binding of VEGF and the intracellular domain is involvedin signal transduction.

VEGF receptor molecules, or fragments thereof, that specifically bind toVEGF can be used in the methods of the invention to bind to andsequester the VEGF protein, thereby preventing it from signaling. Incertain embodiments, the VEGF receptor molecule, or VEGF bindingfragment thereof, is a soluble form, such as sFlt-1. A soluble form ofthe receptor exerts an inhibitory effect on the biological activity ofthe VEGF protein by binding to VEGF, thereby preventing it from bindingto its natural receptors present on the surface of target cells. Alsoincluded are VEGF receptor fusion proteins, examples of which aredescribed below.

In some embodiments, the VEGF antagonist is a chimeric VEGF receptorprotein. A chimeric VEGF receptor protein is a receptor molecule havingamino acid sequences derived from at least two different proteins, atleast one of which is a VEGF receptor protein (e.g., the fit-1 or KDRreceptor), that is capable of binding to and inhibiting the biologicalactivity of VEGF. In certain embodiments, the chimeric VEGF receptorproteins of the invention consist of amino acid sequences derived fromonly two different VEGF receptor molecules; however, amino acidsequences comprising one, two, three, four, five, six, or all sevenIg-like domains from the extracellular ligand-binding region of thefit-1 and/or KDR receptor can be linked to amino acid sequences fromother unrelated proteins, for example, immunoglobulin sequences. Otheramino acid sequences to which Ig-like domains are combined will bereadily apparent to those of ordinary skill in the art. Examples ofchimeric VEGF receptor proteins include, e.g., soluble Flt-1/Fc, KDR/Fc,or FLt-1/KDR/Fc (also known as VEGF Trap). (See for example PCTApplication Publication No. WO97/44453).

A soluble VEGF receptor protein or chimeric VEGF receptor proteins ofthe invention includes VEGF receptor proteins which are not fixed to thesurface of cells via a transmembrane domain. As such, soluble forms ofthe VEGF receptor, including chimeric receptor proteins, while capableof binding to and inactivating VEGF, do not comprise a transmembranedomain and thus generally do not become associated with the cellmembrane of cells in which the molecule is expressed.

In some embodiments, the VEGF antagonist (i, an anti-VEGF antibody, suchas bevacizumab) is administered by gene therapy. See, for example, WO96/07321 published Mar. 14, 1996 concerning the use of gene therapy togenerate intracellular antibodies. There are two major approaches togetting the nucleic acid (optionally contained in a vector) into thepatient's cells; in vivo and ex vivo. For in vivo delivery the nucleicacid is injected directly into the patient, usually at the site wherethe antibody is required. For ex vivo treatment, the patient's cells areremoved, the nucleic acid is introduced into these isolated cells andthe modified cells are administered to the patient either directly or,for example, encapsulated within porous membranes which are implantedinto the patient (see, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187).There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. A commonly used vector for ex vivodelivery of the gene is a retrovirus. The currently preferred in vivonucleic acid transfer techniques include transfection with viral vectors(such as adenovirus, Herpes simplex I virus, or adeno-associated virus)and lipid-based sy stems (useful lipids for lipid-mediated transfer ofthe gene are DOTMA, DOPE and DC-Chol, for example). In some situationsit is desirable to provide the nucleic acid source with an agent thattargets the target cells, such as an antibody specific for a cellsurface membrane protein or the target cell, a ligand for a receptor onthe target cell, etc. Where liposoames are employed, proteins which bindto a cell surface membrane protein associated with endocy tosis may beused for targeting and/or to facilitate uptake. e.g. capsid proteins orfragments thereof tropic for a particular cell type, antibodies forproteins which undergo internalization in cycling, and proteins thattarget intracellular localization and enhance intracellular half-life.The technique of receptor-mediated endocytosis is described, forexample, by Wu et al., J. Biol. Chem. 262:44294432 (1987) and Wagner etal., Proc. Natl. Acad. Sci. USA 87:3410-3414 (1990). For review of thecurrently known gene marking and gene therapy protocols see Anderson etal., Science 256:808-813 (1992). See also WO 93/25673 and the referencescited therein.

Anti-VEGF Antibodies

In some embodiments, the anti-angiogenesis agent is a VEGF antagonist.In some embodiments, the VEGF antagonist is an anti-VEGF antibody. Insome embodiments, the anti-VEGF antibody may be a human or humanizedantibody. In some embodiments, the anti-VEGF antibody may be amonoclonal antibody.

The VEGF antigen to be used for production of VEGF antibodies may be,e.g., the VEGF₁₆₅ molecule as well as other isoforms of VEGF or afragment thereof containing the desired epitope. In one embodiment, thedesired epitope is the one recognized by bevacizumab, which binds to thesame epitope as the monoclonal anti-VEGF antibody A4.6.1 produced byhybridoma ATCC HB 10709 (known as “epitope A.4.6.1” defined herein).Other forms of VEGF useful for generating anti-VEGF antibodies of theinvention will be apparent to those skilled in the art.

Human VEGF was obtained by first screening a cDNA library prepared fromhuman cells, using bovine VEGF cDNA as a hybridization probe. Leung etal. (1989) Science, 246:1306. One cDNA identified thereby encodes a165-amino acid protein having greater than 95% homology to bovine VEGF;this 165-amino acid protein is typically referred to as human VEGF(hVEGF) or VEGF₁₆₅. The mitogenic activity of human VEGF was confirmedby expressing the human VEGF cDNA in mammalian host cells. Mediaconditioned by cells transfected with the human VEGF cDNA promoted theproliferation of capillary endothelial cells, whereas control cells didnot. Leung et al. (1989) Science, supra. Further efforts were undertakento clone and express VEGF via recombinant DNA techniques. (See, e.g.,Ferrara, Laboratory Investigation 72:615-618 (1995), and the referencescited therein).

VEGF is expressed in a variety of tissues as multiple homodimeric forms(121, 145, 165, 189, and 206 amino acids per monomer) resulting fromalternative RNA splicing. VEGF₁₂₁ is a soluble mitogen that does notbind heparin; the longer forms of VEGF bind heparin with progressivelyhigher affinity. The heparin-binding forms of VEGF can be cleaved in thecarboxy terminus by plasmin to release a diffusible form(s) of VEGF.Amino acid sequencing of the carboxy terminal peptide identified afterplasmin cleavage is Arg₁₁₀-Ala₁₁₁. Amino terminal “core” protein, VEGF(1-110) isolated as a homodimer, binds neutralizing monoclonalantibodies (such as the antibodies referred to as 4.6.1 and 3.2E3.1.1)and soluble forms of VEGF receptors with similar affinity compared tothe intact VEGF₁₆₅ homodimer.

Several molecules structurally related to VEGF have also been identifiedrecently, including placenta growth factor (PIGF), VEGF-B, VEGF-C,VEGF-D and VEGF-E. Ferrara and Davis-Smyth (1987) Endocr. Rev., supra;Ogawa et al. J. Biological Chem. 273:31273-31281 (1998); Meyer et al.EMBO J., 18:363-374 (1999). A receptor tyrosine kinase, Flt-4 (VEGFR-3),has been identified as the receptor for VEGF-C and VEGF-D. Joukov et al.EMBO. J. 15:1751 (1996); Lee et al. PNAS USA 93:1988-1992 (1996); Achenet al. (1998) PNAS USA 95:548-553. VEGF-C has been shown to be involvedin the regulation of lymphatic angiogenesis. Jeltsch et al. Science276:1423-1425 (1997).

Two VEGF receptors have been identified, Flt-1 (also called VEGFR-1) andKDR (also called VEGFR-2). Shibuya et al. (1990) Oncogene 8:519-527; deVries et al. (1992) Science 255:989-991; Terman et al. (1992) Biochem.Biophys. Res. Commun. 187:1579-1586. Neuropilin-1 has been shown to be aselective VEGF receptor, able to bind the heparin-binding VEGF isoforms(Soker et al. (1998) Cell 92:735-45).

Anti-VEGF antibodies that are useful in the methods of the inventioninclude any antibody, or antigen binding fragment thereof, that bindwith sufficient affinity and specificity to VEGF and can reduce orinhibit the biological activity of VEGF. An anti-VEGF antibody willusually not bind to other VEGF homologues such as VEGF-B or VEGF-C, norother growth factors such as P1GF, PDGF, or bFGF.

In certain embodiments of the invention, the anti-VEGF antibodiesinclude, but are not limited to, a monoclonal antibody that binds to thesame epitope as the monoclonal anti-VEGF antibody A4.6.1 produced byhybridoma ATCC HB 10709; a recombinant humanized anti-VEGF monoclonalantibody generated according to Presta et al. (1997) Cancer Res.57:4593-4599. In one embodiment, the anti-VEGF antibody is “bevacizumab(BV)”, also known as “rhuMAb VEGF” or “AVASTIN®”. It comprises mutatedhuman IgG1 framework regions and antigen-bindingcomplementarity-determining regions from the murine anti-hVEGFmonoclonal antibody A.4.6.1 that blocks binding of human VEGF to itsreceptors. Approximately 93% of the amino acid sequence of bevacizumab,including most of the framework regions, is derived from human IgG1, andabout 7% of the sequence is derived from the murine antibody A4.6.1.

Bevacizumab (AVASTIN®) was the first anti-angiogenesis therapy approvedby the FDA and is approved for the treatment metastatic colorectalcancer (first- and second-line treatment in combination with intravenous5-FU-based chemotherapy), advanced non-squamous, non-small cell lungcancer (NSCLC) (first-line treatment of unresectable, locally advanced,recurrent or metastatic NSCLC in combination with carboplatin andpaclitaxel) and metastatic HER2-negative breast cancer (previouslyuntreated, metastatic HER2-negative breast cancer in combination withpaclitaxel).

Bevacizumab and other humanized anti-VEGF antibodies are furtherdescribed in U.S. Pat. No. 6,884,879 issued Feb. 26, 2005. Additionalantibodies include the G6 or B20 series antibodies (e.g., G6-31,B20-4.1), as described in PCT Publication No. WO2005/012359, PCTPublication No. WO2005/044853, and U.S. Patent Application 60/991,302,the content of these patent applications are expressly incorporatedherein by reference. For additional antibodies see U.S. Pat. Nos.7,060,269, 6,582,959, 6,703,020; 6,054,297; WO98/45332; WO 96/30046;WO94/10202; EP 0666868B1; U.S. Patent Application Publication Nos.2006009360, 20050186208, 20030206899, 20030190317, 20030203409, and20050112126; and Popkov et al., Journal of Immunological Methods288:149-164 (2004). Other antibodies include those that bind to afunctional epitope on human VEGF comprising of residues F17, M18, D19,Y21, Y25, Q89, 1191, K101, E103, and C104 or, alternatively, comprisingresidues F17, Y21, Q22, Y25, D63, 183 and Q89.

In one embodiment of the invention, the anti-VEGF antibody has a lightchain variable region comprising the following amino acid sequence:

(SEQ ID NO: 214) DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKPGKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ YSTVPWTFGQ GTKVEIKR.;and/or a heavy chain variable region comprising the following amino acidsequence: EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGWINTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYFDVWGQGTLVT VSS (SEQ ID NO:215).

In some embodiments, the anti-VEGF antibody comprises one, two, three,four, five, or six hypervariable region (HVR) sequences of bevacizunmab.In some embodiments, the anti-VEGF antibody comprises one, two, three,four, five, or six hypervariable region (HVR) sequences of selected from(a) HVR-H1 comprising the amino acid sequence of GYTFTNYGMN (SEQ IDNO:216); (b) HVR-H2 comprising the amino acid sequence ofWINTYTGEPTYAADFKR (SEQ ID NO:217); (c) HVR-H3 comprising the amino acidsequence of YPHYYGSSHWYFDV (SEQ ID NO:218); (d) HVR-L1 comprising theamino acid sequence of SASQDISNYLN (SEQ ID NO:219); (e) HVR-L2comprising the amino acid sequence of FTSSLHS (SEQ ID NO:220); and (f)HVR-L3 comprising the amino acid sequence of QQYSTVPWT (SEQ ID NO:221).In some embodiments, the anti-VEGF antibody comprises one, two, three,four, five, or six hypervariable region (HVR) sequences of an antibodydescribed in U.S. Pat. No. 6,884,879. In some embodiments, the anti-VEGFantibody comprises one, two, or three hypervariable region (HVR)sequences of a light chain variable region comprising the followingamino acid sequence: DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKPGKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQGTKVEIKR. (SEQ ID NO:214) and/or one, two, or three hypervariable region(HVR) sequences of a heavy chain variable region comprising thefollowing amino acid sequence: EVQLVESGGG LVQPGGSLRL SCAASGYTFTNYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAEDTAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS (SEQ ID NO:215).

A “G6 series antibody” according to this invention, is an anti-VEGFantibody that is derived from a sequence of a G6 antibody or G6-derivedantibody according to any one of FIGS. 7, 24-26, and 34-35 of PCTPublication No. WO2005/012359, the entire disclosure of which isexpressly incorporated herein by reference. See also PCT Publication No.WO2005/044853, the entire disclosure of which is expressly incorporatedherein by reference. In one embodiment, the G6 series antibody binds toa functional epitope on human VEGF comprising residues F17, Y21, Q22,Y25, D63, 183 and Q89.

A “B20 series antibody” according to this invention is an anti-VEGFantibody that is derived from a sequence of the B20 antibody or aB20-derived antibody according to any one of FIGS. 27-29 of PCTPublication No. WO2005/012359, the entire disclosure of which isexpressly incorporated herein by reference. See also PCT Publication No.WO2005/044853, and U.S. Patent Application 60/991,302, the content ofthese patent applications are expressly incorporated herein byreference. In one embodiment, the B20 series antibody binds to afunctional epitope on human VEGF comprising residues F17, M18, D19, Y21,Y25, Q89, 191, K101, E103, and C104.

A “functional epitope” according to this invention refers to amino acidresidues of an antigen that contribute energetically to the binding ofan antibody. Mutation of any one of the energetically contributingresidues of the antigen (for example, mutation of wild-type VEGF byalanine or homolog mutation) will disrupt the binding of the antibodysuch that the relative affinity ratio (IC50mutant VEGF/IC50wild-typeVEGF) of the antibody will be greater than 5 (see Example 2 ofWO2005/012359). In one embodiment, the relative affinity ratio isdetermined by a solution binding phage displaying ELISA. Briefly,96-well Maxisorp immunoplates (NUNC) are coated overnight at 4° C. withan Fab form of the antibody to be tested at a concentration of 2 μg/mlin PBS, and blocked with PBS, 0.5% BSA, and 0.05% Tween20 (PBT) for 2 hat room temperature. Serial dilutions of phage displaying hVEGF alaninepoint mutants (residues 8-109 form) or wild type hVEGF (8-109) in PBTare first incubated on the Fab-coated plates for 15 min at roomtemperature, and the plates are washed with PBS, 0.05% Tween20 (PBST).The bound phage is detected with an anti-M13 monoclonal antibodyhorseradish peroxidase (Amersham Pharmacia) conjugate diluted 1:5000 inPBT, developed with 3,3′,5,5′-tetramethylbenzidine (TMB, Kirkegaard &Perry Labs, Gaithersburg, Md.) substrate for approximately 5 min,quenched with 1.0 M H3PO4, and read spectrophotometrically at 450 nm.The ratio of IC50 values (IC50,ala/IC50,wt) represents the fold ofreduction in binding affinity (the relative binding affinity).

III. OX40 Binding Agonists

Provided herein are methods treating or delaying progression of cancerin an individual comprising administering to the individual an effectiveamount of an anti-angiogenesis agent and an OX40 binding agonist.

An OX40 binding agonist includes, for example, an OX40 agonist antibody(e.g., an anti-human OX40 agonist antibody), an OX40L agonist fragment,an OX40 oligomeric receptor, and an OX40 immunoadhesin. In someembodiments, the OX40 binding agonist is a trimeric OX40L-Fc protein. Insome embodiments, the OX40 binding agonist is an OX40L agonist fragmentcomprising one or more extracellular domains of OX40L. In someembodiments, the OX40 agonist antibody is a full-length human IgG1antibody. Any of the OX40 binding agonists (e.g., anti-human OX40agonist antibodies) described herein may be used in any of the methods,uses, and/or kits described herein.

In some embodiments, the OX40 agonist antibody increases CD4+ effector Tcell proliferation and/or increases cytokine production by the CD4+effector T cell as compared to proliferation and/or cytokine productionprior to treatment with the OX40 agonist antibody. In some embodiments,the cytokine is IFN-γ.

In some embodiments, the OX40 agonist antibody increases memory T cellproliferation and/or increasing cytokine production by the memory cell.In some embodiments, the cytokine is IFN-γ.

In some embodiments, the OX40 agonist antibody inhibits Treg suppressionof effector T cell function. In some embodiments, effector T cellfunction is effector T cell proliferation and/or cytokine production. Insome embodiments, the effector T cell is a CD4+ effector T cell.

In some embodiments, the OX40 agonist antibody increases OX40 signaltransduction in a target cell that expresses OX40. In some embodiments,OX40 signal transduction is detected by monitoring NFkB downstreamsignaling.

In some embodiments, the anti-human OX40 agonist antibody is a depletinganti-human OX40 antibody (e.g., depletes cells that express human OX40).In some embodiments, the OX40 agonist antibody depletes cells thatexpress human OX40 in vitro. In some embodiments, the human OX40expressing cells are CD4+ effector T cells. In some embodiments, thehuman OX40 expressing cells are Treg cells. In some embodiments,depleting is by ADCC and/or phagocytosis. In some embodiments, theantibody mediates ADCC by binding FcγR expressed by a human effectorcell and activating the human effector cell function. In someembodiments, the antibody mediates phagocytosis by binding FcγRexpressed by a human effector cell and activating the human effectorcell function. Exemplary human effector cells include, e.g., macrophage,natural killer (NK) cells, monocytes, neutrophils. In some embodiments,the human effector cell is macrophage.

In some embodiments, the anti-human OX40 agonist antibody has afunctional Fc region. In some embodiments, effector function of afunctional Fc region is ADCC. In some embodiments, effector function ofa functional Fc region is phagocytosis. In some embodiments, effectorfunction of a functional Fc region is ADCC and phagocytosis. In someembodiments, the Fc region is human IgG1. In some embodiments, the Fcregion is human IgG4.

In some embodiments, the anti-human OX40 agonist antibody binds humanOX40 with an affinity of less than or equal to about 0.45 nM. In someembodiments, the anti-human OX40 antibody binds human OX40 with anaffinity of less than or equal to about 0.4 nM. In some embodiments, theanti-human OX40 antibody binds human OX40 with an affinity of less thanor equal to about 0.5 nM. In some embodiments, the binding affinity isdetermined using radioimmunoassay.

In some embodiments, the OX40 binding agonist is an OX40 agonistantibody that binds human OX40. In some embodiments, the OX40 agonistantibody binds human OX40 with an affinity of less than or equal toabout 1 nM. In some embodiments, the anti-human OX40 agonist antibodybinds human OX40 with an affinity of less than or equal to about 0.45nM. In some embodiments, the anti-human OX40 antibody binds human OX40with an affinity of less than or equal to about 0.4 nM. In someembodiments, the anti-human OX40 antibody binds human OX40 with anaffinity of less than or equal to about 0.5 nM. In some embodiments, thebinding affinity is determined using radioimmunoassay.

In some embodiments, the anti-human OX40 agonist antibody binds humanOX40 and cynomolgus OX40. In some embodiments, binding is determinedusing a FACS assay. In some embodiments, binding to human OX40 has anEC50 of about 0.2 ug/ml. In some embodiments, binding to human OX40 hasan EC50 of about 0.3 ug/ml or lower. In some embodiments, binding tocynomolgus OX40 has an EC50 of about 1.5 ug/ml. In some embodiments,binding to cynomolgus OX40 has an EC50 of about 1.4 ug/ml.

In some embodiments, the anti-human OX40 agonist antibody does not bindto rat OX40 or mouse OX40.

In some embodiments, the anti-human OX40 agonist antibody is a depletinganti-human OX40 antibody (e.g., depletes cells that express human OX40).In some embodiments, the human OX40 expressing cells are CD4+ effector Tcells. In some embodiments, the human OX40 expressing cells are Tregcells. In some embodiments, depleting is by ADCC and/or phagocytosis. Insome embodiments, the antibody mediates ADCC by binding FcγR expressedby a human effector cell and activating the human effector cellfunction. In some embodiments, the antibody mediates phagocytosis bybinding FcγR expressed by a human effector cell and activating the humaneffector cell function. Exemplary human effector cells include, e.g.,macrophage, natural killer (NK) cells, monocytes, neutrophils. In someembodiments, the human effector cell is macrophage. In some embodiments,the human effector cell is NK cells. In some embodiments, depletion isnot by apoptosis.

In some embodiments, the anti-human OX40 agonist antibody has afunctional Fc region. In some embodiments, effector function of afunctional Fc region is ADCC. In some embodiments, effector function ofa functional Fc region is phagocytosis. In some embodiments, effectorfunction of a functional Fc region is ADCC and phagocytosis. In someembodiments, the Fc region is human IgG1. In some embodiments, the Fcregion is human IgG4.

In some embodiments, the anti-human OX40 agonist antibody does notinduce apoptosis in OX40-expressing cells (e.g., Treg). In someembodiments, apoptosis is assayed using an antibody concentration of 30ug/ml, e.g., by determining whether apoptosis has occurred using annexinV and proprodium iodide stained Treg.

In some embodiments, the anti-human OX40 agonist antibody enhances CD4+effector T cell function, for example, by increasing CD4+ effector Tcell proliferation and/or increasing gamma interferon production by theCD4+ effector T cell (for example, as compared to proliferation and/orcytokine production prior to treatment with anti-human OX40 agonistantibody). In some embodiments, the cytokine is gamma interferon. Insome embodiments, the anti-human OX40 agonist antibody increases numberof intratumoral (infiltrating) CD4+ effector T cells (e.g., total numberof CD4+ effector T cells, or e.g., percentage of CD4+ cells in CD45+cells), e.g., as compared to number of intratumoral (infiltrating) CD4+T cells prior to treatment with anti-human OX40 agonist antibody. Insome embodiments, the anti-human OX40 agonist antibody increases numberof intratumoral (infiltrating) CD4+ effector T cells that express gammainterferon (e.g., total gamma interferon expressing CD4+ cells, or e.g.,percentage of gamma interferon expressing CD4+ cells in total CD4+cells), e.g., as compared to number of intratumoral (infiltrating) CD4+T cells that express gamma interferon prior to treatment with anti-humanOX40 agonist antibody.

In some embodiments, the anti-human OX40 agonist antibody increasesnumber of intratumoral (infiltrating) CD8+ effector T cells (e.g., totalnumber of CD8+ effector T cells, or e.g., percentage of CD8+ in CD45+cells), e.g., as compared to number of intratumoral (infiltrating) CD8+Teffector cells prior to treatment with anti-human OX40 agonist antibody.In some embodiments, the anti-human OX40 agonist antibody increasesnumber of intratumoral (infiltrating) CD8+ effector T cells that expressgamma interferon (e.g., percentage of CD8+ cells that express gammainterferon in total CD8+ cells), e.g., compared to number ofintratumoral (infiltrating) CD8+ T cells that express gamma interferonprior to treatment with anti-human OX40 agonist antibody.

In some embodiments, the anti-human OX40 agonist antibody enhancesmemory T cell function, for example by increasing memory T cellproliferation and/or increasing cytokine production by the memory cell.In some embodiments, the cytokine is gamma interferon.

In some embodiments, the anti-human OX40 agonist antibody inhibits Tregfunction, for example, by decreasing Treg suppression of effector T cellfunction (e.g., effector T cell proliferation and/or effector T cellcytokine secretion). In some embodiments, the effector T cell is a CD4+effector T cell. In some embodiments, the anti-human OX40 agonistantibody reduces the number of intratumoral (infiltrating) Treg (e.g.,total number of Treg or e.g., percentage of Fox3p+ cells in CD4+ cells).

In some embodiments, the anti-human OX40 agonist antibody is engineeredto increase effector function (e.g., compared to effector function in awild-type IgG1). In some embodiments, the antibody has increased bindingto a Fc receptor. In some embodiments, the antibody lacks fucoseattached (directly or indirectly) to the Fc region. For example, theamount of fucose in such antibody may be from 1% to 80%, from 1% to 65%,from 5% to 65% or from 20% to 40%. In some embodiments, the Fc regioncomprises bisected oligosaccharides, e.g., in which a biantennaryoligosaccharide attached to the Fc region of the antibody is bisected byGlcNAc. In some embodiments, the antibody comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, the anti-human OX40 agonist antibody increases OX40signal transduction in a target cell that expresses OX40. In someembodiments, OX40 signal transduction is detected by monitoring NFkBdownstream signaling.

In some embodiments, the anti-human OX40 agonist antibody is stableafter treatment at 40° C. for two weeks.

In some embodiments, the anti-human OX40 agonist antibody binds humaneffector cells, e.g., binds FcγR (e.g., an activating FcγR) expressed byhuman effector cells. In some embodiments, the human effector cellperforms (is capable of performing) ADCC effector function. In someembodiments, the human effector cell performs (is capable of performing)phagocytosis effector function.

In some embodiments, the anti-human OX40 agonist antibody comprising avariant IgG1 Fc polypeptide comprising a mutation that eliminatesbinding to human effector cells (e.g., a DANA mutation) has diminishedactivity (e.g., CD4+ effector T cell function, e.g., proliferation),relative to anti-human OX40 agonist antibody comprising native sequenceIgG1 Fc portion. In some embodiment, the anti-human OX40 agonistantibody comprising a variant IgG1 Fc polypeptide comprising a mutationthat eliminates binding to human effector cells (e.g., a DANA mutation)does not possess substantial activity (e.g., CD4+ effector T cellfunction, e.g., proliferation).

In some embodiments, antibody cross-linking is required for anti-humanOX40 agonist antibody function. In some embodiments, function isstimulation of CD4+ effector T cell proliferation. In some embodiments,antibody cross-linking is determined by providing anti-human OX40agonist antibody adhered on a solid surface (e.g., a cell cultureplate). In some embodiments, antibody cross-linking is determined byintroducing a mutation in the antibody's IgG1 Fc portion (e.g., a DANAmutation) and testing function of the mutant antibody.

In some embodiments, the anti-human OX40 agonist antibody competes forbinding to human OX40 with OX40L. In some embodiments, addition of OX40Ldoes not enhance anti-human OX40 antibody function in an in vitro assay.

According to another embodiment, the anti-human OX40 agonist antibodiesinclude any one, any combination, or all of the following properties:(1) binds human OX40 with an affinity of less than or equal to about0.45 nM, in some embodiments, binds human OX40 with an affinity of lessthan or equal to about 0.4 nM, in some embodiments, binds human OX40with an affinity of less than or equal to about 0.5 nM, in someembodiments, the binding affinity is determined using radioimmunoassay;(2) binds human OX40 and cynomolgus OX40, in some embodiments, bindingis determined using a FACS assay, (3) binds human OX40 with an EC50 ofabout 0.2 ug/ml, in some embodiments, binds to human OX40 has an EC50 ofabout 0.3 ug/ml or lower, in some embodiments, binds to cynomolgus OX40with an EC50 of about 1.5 ug/ml, in some embodiments, binds tocynomolgus OX40 has an EC50 of about 1.4 ug/ml, (4) does notsubstantially bind to rat OX40 or mouse OX40, (6) is a depletinganti-human OX40 antibody (e.g., depletes cells that express human OX40),in some embodiments, the cells are CD4+ effector T cells and/or Tregcells, (7) enhances CD4+ effector T cell function, for example, byincreasing CD4+ effector T cell proliferation and/or increasing gammainterferon production by the CD4+ effector T cell (for example, ascompared to proliferation and/or cytokine production prior to treatmentwith anti-human OX40 agonist antibody), (8) enhances memory T cellfunction, for example by increasing memory T cell proliferation and/orincreasing cytokine production by the memory cell, (9) inhibits Tregfunction, for example, by decreasing Treg suppression of effector T cellfunction (e.g., effector T cell proliferation and/or effector T cellcytokine secretion). In some embodiments, the effector T cell is a CD4+effector T cell, (10) increases OX40 signal transduction in a targetcell that expresses OX40 (in some embodiments, OX40 signal transductionis detected by monitoring NFkB downstream signaling), (11) is stableafter treatment at 40° C. for two weeks, (12) binds human effectorcells, e.g., binds FcγR expressed by human effector cells, (13)anti-human OX40 agonist antibody comprising a variant IgG1 Fcpolypeptide comprising a mutation that eliminates binding to humaneffector cells (e.g., N297G) has diminished activity (e.g., CD4+effector T cell function, e.g., proliferation), relative to anti-humanOX40 agonist antibody comprising native sequence IgG1 Fc portion, insome embodiment, the anti-human OX40 agonist antibody comprising avariant IgG1 Fc polypeptide comprising a mutation that eliminatesbinding to human effector cells (e.g., N297G) does not possesssubstantial activity (e.g., CD4+ effector T cell function, e.g.,proliferation), (14) antibody cross-linking (e.g., by Fc receptorbinding) is required for anti-human OX40 agonist antibody function.

In one aspect, the invention provides an anti-human OX40 agonistantibody comprising at least one, two, three, four, five, or six HVRsselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3; (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In one aspect, the invention provides an anti-human OX40 agonistantibody comprising at least one, at least two, or all three VH HVRsequences selected from (a) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:3; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4.In one embodiment, the antibody comprises HVR-H3 comprising the aminoacid sequence of SEQ ID NO:4. In another embodiment, the antibodycomprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:4 andHVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In a furtherembodiment, the antibody comprises HVR-H3 comprising the amino acidsequence of SEQ ID NO:4, HVR-L3 comprising the amino acid sequence ofSEQ ID NO:7, and HVR-H2 comprising the amino acid sequence of SEQ IDNO:3. In a further embodiment, the antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprisingthe amino acid sequence of SEQ ID NO:3; and (c) HVR-H3 comprising theamino acid sequence of SEQ ID NO:4.

In another aspect, the invention provides an anti-human OX40 agonistantibody comprising at least one, at least two, or all three VL HVRsequences selected from (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:6; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.In one embodiment, the antibody comprises (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino acidsequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7.

In another aspect, an anti-human OX40 agonist antibody of the inventioncomprises (a) a VH domain comprising at least one, at least two, or allthree VH HVR sequences selected from (i) HVR-H1 comprising the aminoacid sequence of SEQ ID NO:2, (ii) HVR-H2 comprising the amino acidsequence of SEQ ID NO:3, and (iii) HVR-H3 comprising an amino acidsequence selected from SEQ ID NO:4; and (b) a VL domain comprising atleast one, at least two, or all three VL HVR sequences selected from (i)HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO:6, and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:7.

In another aspect, the invention provides an anti-human OX40 agonistantibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising anamino acid sequence selected from SEQ ID NO:7.

In one aspect, the invention provides an anti-human OX40 agonistantibody comprising at least one, two, three, four, five, or six HVRsselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3; (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:26.

In another embodiment, the antibody comprises HVR-H3 comprising theamino acid sequence of SEQ ID NO:4 and HVR-L3 comprising the amino acidsequence of SEQ ID NO:26. In a further embodiment, the antibodycomprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:4,HVR-L3 comprising the amino acid sequence of SEQ ID NO:26, and HVR-H2comprising the amino acid sequence of SEQ ID NO:3.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:2, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:3, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:4; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:26.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:2; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:3; (c) HVR-H3 comprisingthe amino acid sequence of SEQ ID NO:4; (d) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acidsequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acidsequence selected from SEQ ID NO:26.

In one aspect, the invention provides an anti-human OX40 agonistantibody comprising at least one, two, three, four, five, or six HVRsselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3; (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:27.

In another embodiment, the antibody comprises HVR-H3 comprising theamino acid sequence of SEQ ID NO:4 and HVR-L3 comprising the amino acidsequence of SEQ ID NO:27. In a further embodiment, the antibodycomprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:4,HVR-L3 comprising the amino acid sequence of SEQ ID NO:27, and HVR-H2comprising the amino acid sequence of SEQ ID NO:3.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:2, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:3, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:4; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:27.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:2; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:3; (c) HVR-H3 comprisingthe amino acid sequence of SEQ ID NO:4; (d) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acidsequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acidsequence selected from SEQ ID NO:27.

In one aspect, the invention provides an anti-human OX40 agonistantibody comprising at least one, two, three, four, five, or six HVRsselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:2, 8 or 9; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:3, 10, 11, 12, 13 or 14; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:4, 15, or 19; (d) HVR-L1 comprising the amino acidsequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequenceof SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid sequence of SEQID NO:7, 22, 23, 24, 25, 26, 27, or 28.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2, 8 or 9; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3, 10, 11, 12,13 or 14; and (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 4, 15, or 19. In one embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO: 4, 15, or 19. Inanother embodiment, the antibody comprises HVR-H3 comprising the aminoacid sequence of SEQ ID NO:4, 15, or 19 and HVR-L3 comprising the aminoacid sequence of SEQ ID NO: 7, 22, 23, 24, 25, 26, 27, or 28. In afurther embodiment, the antibody comprises HVR-H3 comprising the aminoacid sequence of SEQ ID NO: 4, 15, or 19, HVR-L3 comprising the aminoacid sequence of SEQ ID NO: 7, 22, 23, 24, 25, 26, 27, or 28, and HVR-H2comprising the amino acid sequence of SEQ ID NO: 3, 10, 11, 12, 13 or14. In a further embodiment, the antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 2, 8 or 9; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 3, 10, 11, 12, 13 or14; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4,15, or 19.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 5; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 7, 22, 23, 24, 25, 26,27, or 28. In one embodiment, the antibody comprises (a) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 7, 22, 23, 24, 25, 26, 27, or 28.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 2, 8 or 9, (ii) HVR-H2 comprising the amino acid sequence ofSEQ ID NO: 3, 10, 11, 12, 13 or 14, and (iii) HVR-H3 comprising an aminoacid sequence selected from SEQ ID NO: 4, 15, or 19; and (b) a VL domaincomprising at least one, at least two, or all three VL HVR sequencesselected from (i) HVR-L1 comprising the amino acid sequence of SEQ IDNO:5, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 7, 22, 23,24, 25, 26, 27, or 28.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2, 8 or 9; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3, 10, 11, 12,13 or 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4,15, or 19; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f)HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 7, 22,23, 24, 25, 26, 27, or 28.

In one aspect, the invention provides an anti-human OX40 agonistantibody comprising at least one, two, three, four, five, or six HVRsselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:172; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:173;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 174; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:175. In some embodiment,HVR-H2 is not DMYPDAAAASYNQKFRE (SEQ ID NO:230).In some embodiments,HVR-H3 is not APRWAAAA (SEQ ID NO:231). In some embodiments, HVR-L3 isnot QAAAAAAAT (SEQ ID NO:232).

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:172; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 173; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 174. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO: 174. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO: 174 and HVR-L3comprising the amino acid sequence of SEQ ID NO: 175. In a furtherembodiment, the antibody comprises HVR-H3 comprising the amino acidsequence of SEQ ID NO: 174, HVR-L3 comprising the amino acid sequence ofSEQ ID NO: 175, and HVR-H2 comprising the amino acid sequence of SEQ IDNO:173. In a further embodiment, the antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 172; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 173; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 174. In someembodiment, HVR-H2 is not DMYPDAAAASYNQKFRE (SEQ ID NO:230).In someembodiments, HVR-H3 is not APRWAAAA (SEQ ID NO:231). In someembodiments, HVR-L3 is not QAAAAAAAT (SEQ ID NO:232).

In another aspect, the invention provides an antibody comprising (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 175. In someembodiments, HVR-L3 is not QAAAAAAAT (SEQ ID NO:232).

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 172, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:173, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:174; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:175.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 172; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 173; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 174; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising anamino acid sequence selected from SEQ ID NO:175. In some embodiment,HVR-H2 is not DMYPDAAAASYNQKFRE (SEQ ID NO:230).In some embodiments,HVR-H3 is not APRWAAAA (SEQ ID NO:231). In some embodiments, HVR-L3 isnot QAAAAAAAT (SEQ ID NO:232).

All possible combinations of the above substitutions are encompassed bythe consensus sequences of SEQ ID NO:172, 173, 174 and 175.

In one aspect, the invention provides an anti-human OX40 agonistantibody comprising at least one, two, three, four, five, or six HVRsselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:39; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:42.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:30; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:33. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:33. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:33 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:42. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:33, HVR-L3 comprising the amino acid sequence of SEQ ID NO:42, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:30. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:30; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:33.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:39; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:42. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:37; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:39; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:42.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:29, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:30, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:33; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:37, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:39, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:42.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:30; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:33; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:37; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:39; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:42.

In one aspect, the invention provides an anti-human OX40 agonistantibody comprising at least one, two, three, four, five, or six HVRsselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:40; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:42.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:40; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:42. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:37; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:40; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:42.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:29, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:30, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:33; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:37, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:40, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:42.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:30; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:33; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:37; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:40; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:42.

In one aspect, the invention provides an anti-human OX40 agonistantibody comprising at least one, two, three, four, five, or six HVRsselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30,31, or 32; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO:33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, 40 or 41;and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42, 43,or 44.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 30, 31, or 32; and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:33. In anotherembodiment, the antibody comprises HVR-H3 comprising the amino acidsequence of SEQ ID NO:33 and HVR-L3 comprising the amino acid sequenceof SEQ ID NO: 42, 43, or 44. In a further embodiment, the antibodycomprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:33,HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42, 43, or 44,and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 39, 40 or41. In a further embodiment, the antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:30, 31, or 32; and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:33.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 39, 40 or 41; and (c)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42, 43, or 44.In one embodiment, the antibody comprises (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO:37; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 39, 40 or 41; and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 42, 43, or 44.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:29, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 30, 31, or 32, and (iii) HVR-H3 comprising an amino acid sequenceselected from SEQ ID NO:33; and (b) a VL domain comprising at least one,at least two, or all three VL HVR sequences selected from (i) HVR-L1comprising the amino acid sequence of SEQ ID NO:37, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO: 39, 40 or 41, and (c)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42, 43, or 44.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 30, 31, or 32; (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:37; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 39, 40 or 41; and (f)HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 42,43, or 44.

In one aspect, the invention provides an anti-human OX40 agonistantibody comprising at least one, two, three, four, five, or six HVRsselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 175;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 177; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 178.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:175; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:33. In anotherembodiment, the antibody comprises HVR-H3 comprising the amino acidsequence of SEQ ID NO:33 and HVR-L3 comprising the amino acid sequenceof SEQ ID NO: 177. In a further embodiment, the antibody comprisesHVR-H3 comprising the amino acid sequence of SEQ ID NO:33, HVR-L3comprising the amino acid sequence of SEQ ID NO:178, and HVR-H2comprising the amino acid sequence of SEQ ID NO:176. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 176; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:33.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 177; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 177. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:37; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO: 177; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:178.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:29, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:176, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:33; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:37, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 177, and (c) HVR-L3 comprising the aminoacid sequence of SEQ ID NO:178.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 176; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:33; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:37; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 177; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:178.

In any of the above embodiments, an anti-OX40 agonist antibody ishumanized. In one embodiment, an anti-OX40 antibody comprises HVRs as inany of the above embodiments or for any of the embodiments in FIG. 11,and further comprises an acceptor human framework, e.g. a humanimmunoglobulin framework or a human consensus framework. In anotherembodiment, an anti-OX40 antibody comprises HVRs as in any of the aboveembodiments, and further comprises a VH and/or VL comprising an FRsequence shown in FIG. 11.

In another aspect, an anti-human OX40 agonist antibody comprises a heavychain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO:56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 108, 114, 116, 233, or234. In certain embodiments, a VH sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-human OX40agonist antibody comprising that sequence retains the ability to bind toOX40. In certain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO:56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,108, 114, 116, 233, or 234. In certain embodiments, substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs). Optionally, the anti-human OX40 agonist antibody comprises the VHsequence in SEQ ID NO: SEQ ID NO:56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 108, 114, 116, 233,or 234, including post-translational modifications of that sequence. Ina particular embodiment, the VH comprises one, two or three HVRsselected from: (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:2, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4.

In another aspect, an anti-human OX40 agonist antibody is provided,wherein the antibody comprises a light chain variable domain (VL) havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO:57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 109, 115 or 117. In certain embodiments, a VL sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identitycontains substitutions (e.g., conservative substitutions), insertions,or deletions relative to the reference sequence, but an anti-human OX40agonist antibody comprising that sequence retains the ability to bind toOX40. In certain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO: 57, 59, 61, 63, 65,67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,109, 115 or 117. In certain embodiments, the substitutions, insertions,or deletions occur in regions outside the HVRs (i.e., in the FRs).Optionally, the anti-human OX40 agonist antibody comprises the VLsequence in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 109, 115 or 117, includingpost-translational modifications of that sequence. In a particularembodiment, the VL comprises one, two or three HVRs selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:7.

In another aspect, an anti-human OX40 agonist antibody comprises a heavychain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO:56. In certain embodiments, a VH sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-human OX40 agonist antibody comprising that sequence retains theability to bind to OX40. In certain embodiments, a total of 1 to 10amino acids have been substituted, inserted and/or deleted in SEQ IDNO:56. In certain embodiments, substitutions, insertions, or deletionsoccur in regions outside the HVRs (i.e., in the FRs). Optionally, theanti-human OX40 agonist antibody comprises the VH sequence in SEQ IDNO:56, including post-translational modifications of that sequence. In aparticular embodiment, the VH comprises one, two or three HVRs selectedfrom: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:4.

In another aspect, an anti-human OX40 agonist antibody is provided,wherein the antibody comprises a light chain variable domain (VL) havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO:57. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-human OX40 agonist antibody comprisingthat sequence retains the ability to bind to OX40. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 57. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-human OX40 agonistantibody comprises the VL sequence in SEQ ID NO: 57, includingpost-translational modifications of that sequence. In a particularembodiment, the VL comprises one, two or three HVRs selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:7.

In another aspect, an anti-human OX40 agonist antibody comprises a heavychain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 179. In certain embodiments, a VH sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-human OX40 agonist antibody comprising that sequence retains theability to bind to OX40. In certain embodiments, a total of 1 to 10amino acids have been substituted, inserted and/or deleted in SEQ ID NO:179. In certain embodiments, substitutions, insertions, or deletionsoccur in regions outside the HVRs (i.e., in the FRs). Optionally, theanti-human OX40 agonist antibody comprises the VH sequence in SEQ ID NO:179, including post-translational modifications of that sequence. In aparticular embodiment, the VH comprises one, two or three HVRs selectedfrom: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:4.

In another aspect, an anti-human OX40 agonist antibody is provided,wherein the antibody comprises a light chain variable domain (VL) havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 180. Incertain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-human OX40 agonist antibody comprisingthat sequence retains the ability to bind to OX40. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 180. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-human OX40 agonistantibody comprises the VL sequence in SEQ ID NO: 180, includingpost-translational modifications of that sequence. In a particularembodiment, the VL comprises one, two or three HVRs selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:7.

In another aspect, an anti-human OX40 agonist antibody comprises a heavychain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO:94. In certain embodiments, a VH sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-human OX40 agonist antibody comprising that sequence retains theability to bind to OX40. In certain embodiments, a total of 1 to 10amino acids have been substituted, inserted and/or deleted in SEQ IDNO:94. In certain embodiments, substitutions, insertions, or deletionsoccur in regions outside the HVRs (i.e., in the FRs). Optionally, theanti-human OX40 agonist antibody comprises the VH sequence in SEQ IDNO:94, including post-translational modifications of that sequence. In aparticular embodiment, the VH comprises one, two or three HVRs selectedfrom: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:4.

In another aspect, an anti-human OX40 agonist antibody is provided,wherein the antibody comprises a light chain variable domain (VL) havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO:95. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-human OX40 agonist antibody comprisingthat sequence retains the ability to bind to OX40. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO:95. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-human OX40 agonistantibody comprises the VL sequence in SEQ ID NO:95, includingpost-translational modifications of that sequence. In a particularembodiment, the VL comprises one, two or three HVRs selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:26.

In another aspect, an anti-human OX40 agonist antibody comprises a heavychain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO:96. In certain embodiments, a VH sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-human OX40 agonist antibody comprising that sequence retains theability to bind to OX40. In certain embodiments, a total of 1 to 10amino acids have been substituted, inserted and/or deleted in SEQ IDNO:96. In certain embodiments, substitutions, insertions, or deletionsoccur in regions outside the HVRs (i.e., in the FRs). Optionally, theanti-human OX40 agonist antibody comprises the VH sequence in SEQ IDNO:96, including post-translational modifications of that sequence. In aparticular embodiment, the VH comprises one, two or three HVRs selectedfrom: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:4.

In another aspect, an anti-human OX40 agonist antibody is provided,wherein the antibody comprises a light chain variable domain (VL) havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO:97. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-human OX40 agonist antibody comprisingthat sequence retains the ability to bind to OX40. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO:97. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-human OX40 agonistantibody comprises the VL sequence in SEQ ID NO:97, includingpost-translational modifications of that sequence. In a particularembodiment, the VL comprises one, two or three HVRs selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:27.

In another aspect, an anti-human OX40 agonist antibody comprises a heavychain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 118, 120, 122, 124, 126, 128, 130, 132, 134,136, 138, 140, 142, 144, 146, 148. In certain embodiments, a VH sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-human OX40 agonist antibody comprising that sequence retains theability to bind to OX40. In certain embodiments, a total of 1 to 10amino acids have been substituted, inserted and/or deleted in SEQ ID NO:118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148. In certain embodiments, substitutions, insertions, ordeletions occur in regions outside the HVRs (i.e., in the FRs).Optionally, the anti-human OX40 agonist antibody comprises the VHsequence in SEQ ID NO: 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, including post-translational modificationsof that sequence. In a particular embodiment, the VH comprises one, twoor three HVRs selected from: (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 29, (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:30, and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:33.

In another aspect, an anti-human OX40 agonist antibody is provided,wherein the antibody comprises a light chain variable domain (VL) havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 119, 121,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149. Incertain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-human OX40 agonist antibody comprisingthat sequence retains the ability to bind to OX40. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 119, 121, 123, 125, 127, 129, 131,133, 135, 137, 139, 141, 143, 145, 147, 149. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-human OX40 agonistantibody comprises the VL sequence in SEQ ID NO: 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, includingpost-translational modifications of that sequence. In a particularembodiment, the VL comprises one, two or three HVRs selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:39; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:42.

In one embodiment, the antibody comprises the VH and VL sequences in SEQID NO:56 and SEQ ID NO:57, respectively, including post-translationalmodifications of those sequences. In one embodiment, the antibodycomprises the VH and VL sequences in SEQ ID NO:58 and SEQ ID NO:59,respectively, including post-translational modifications of thosesequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:60 and SEQ ID NO:61, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:62 and SEQID NO:63, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:64 and SEQ ID NO:65, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:66 and SEQID NO:67, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:68 and SEQ ID NO:69, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:70 and SEQID NO:71, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:72 and SEQ ID NO:73, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:74 and SEQID NO:75, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:76 and SEQ ID NO:77, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:78 and SEQID NO:79, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:80 and SEQ ID NO:81, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:82 and SEQID NO:83, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:84 and SEQ ID NO:85, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:86 and SEQID NO:87, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:88 and SEQ ID NO:89, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:90 and SEQID NO:91, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:92 and SEQ ID NO:93, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:94 and SEQID NO:95, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:96 and SEQ ID NO:97, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:98 and SEQID NO:99, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:100 and SEQ ID NO: 101, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO: 108 and SEQID NO: 109, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO: 114 and SEQ ID NO: 115, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO: 116 and SEQID NO: 117, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:233 and SEQ ID NO:65, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:234 and SEQID NO:69, respectively, including post-translational modifications ofthose sequences.

In one embodiment, the antibody comprises the VH and VL sequences in SEQID NO: 118 and SEQ ID NO: 119, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO: 120 and SEQID NO:121, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO: 122 and SEQ ID NO: 123, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO: 124 and SEQID NO: 125, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO: 126 and SEQ ID NO: 127, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO: 128 and SEQID NO: 129, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO: 130 and SEQ ID NO:131, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO: 132 and SEQID NO: 133, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO: 134 and SEQ ID NO:135, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO: 136 and SEQID NO:137, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO: 138 and SEQ ID NO: 139, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO: 140 and SEQID NO:141, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO: 142 and SEQ ID NO: 143, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO: 144 and SEQID NO: 145, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO: 146 and SEQ ID NO: 147, respectively, includingpost-translational modifications of those sequences.

In another aspect, an anti-human OX40 agonist antibody is provided,wherein the antibody comprises a VH as in any of the embodimentsprovided above, and a VL as in any of the embodiments provided above.

In a further aspect, the invention provides an antibody that binds tothe same epitope as an anti-human OX40 antibody provided herein. In someembodiments, the antibody is an anti-human OX40 agonist antibody.

In some embodiments, the anti-human OX40 agonist antibody is a human orhumanized antibody. In some embodiments, the OX40 binding agonist (e.g.,an OX40 agonist antibody) is not MEDI6383. In some embodiments, the OX40binding agonist (e.g., an OX40 agonist antibody) is not MEDI0562.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in U.S. Pat. No. 7,550,140, which isincorporated herein by reference in its entirety. In some embodiments,the anti-human OX40 agonist antibody comprises a heavy chain comprisingthe sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYTMNWVRQAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRYSQVHYALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:183) and/or a light chain comprising thesequence ofDIVMTQSPDSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKAGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYNHPTTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 184). In some embodiments,the antibody comprises at least one, two, three, four, five, or sixhypervariable region (HVR) sequences of antibody 008 as described inU.S. Pat. No. 7,550,140. In some embodiments, the antibody comprises aheavy chain variable region sequence and/or a light chain variableregion sequence of antibody 008 as described in U.S. Pat. No. 7,550,140.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in U.S. Pat. No. 7,550,140. In someembodiments, the anti-human OX40 agonist antibody comprises the sequenceof DIQMTQSPDSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKAGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYNHPTTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 185). In some embodiments,the antibody comprises at least one, two, three, four, five, or sixhypervariable region (HVR) sequences of antibody SC02008 as described inU.S. Pat. No. 7,550,140. In some embodiments, the antibody comprises aheavy chain variable region sequence and/or a light chain variableregion sequence of antibody SC02008 as described in U.S. Pat. No.7,550,140.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in U.S. Pat. No. 7,550,140. In someembodiments, the anti-human OX40 agonist antibody comprises a heavychain comprising the sequence of EVQLVESGGGLVHPGGSLRLSCAGSGFTFSSYAMHWVRQAPGKGLEWVSAIGTGGGTYYA DSVMGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYDNVMGLYWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:186) and/or a light chain comprising thesequence ofEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:187). In some embodiments, theantibody comprises at least one, two, three, four, five, or sixhypervariable region (HVR) sequences of antibody 023 as described inU.S. Pat. No. 7,550,140. In some embodiments, the antibody comprises aheavy chain variable region sequence and/or a light chain variableregion sequence of antibody 023 as described in U.S. Pat. No. 7,550,140.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in U.S. Pat. No. 7,960,515, which isincorporated herein by reference in its entirety. In some embodiments,the anti-human OX40 agonist antibody comprises a heavy chain variableregion comprising the sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIDYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARESGWYLFDYWGQGTLVTVSS (SEQ ID NO:188) and/or a light chain variable region comprising the sequence ofDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGGGTKVEIK (SEQ ID NO:189). In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody 11D4 asdescribed in U.S. Pat. No. 7,960,515. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody 11D4 as described in U.S. Pat. No.7,960,515.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in U.S. Pat. No. 7,960,515. In someembodiments, the anti-human OX40 agonist antibody comprises a heavychain variable region comprising the sequence of EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYA DSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDQSTADYYFYYGMDVWGQGTTVT VSS (SEQ IDNO: 190) and/or a light chain variable region comprising the sequence ofEIVVTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK (SEQ ID NO: 191). In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody 18D8 asdescribed in U.S. Pat. No. 7,960,515. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody 18D8 as described in U.S. Pat. No.7,960,515.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in WO 2012/027328, which is incorporatedherein by reference in its entirety. In some embodiments, the anti-humanOX40 agonist antibody comprises a heavy chain variable region comprisingthe sequence ofQVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQGLKWMGWINTETGEPTYADDFKGRFVFSLDTSVSTAYLQI SSLKAEDTAVYYCANPYYDYVSYYAMDYWGQGTTVTVS S (SEQ IDNO: 192) and/or a light chain variable region comprising the sequence ofDIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYLYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPRTFGQGTKLEIK (SEQ ID NO:193). In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody hu106-222as described in WO 2012/027328. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody hu106-222 as described in WO2012/027328.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in WO 2012/027328. In some embodiments, theanti-human OX40 agonist antibody comprises a heavy chain variable regioncomprising the sequence of EVQLVESGGGLVQPGGSLRLSCAASEYEFPSHDMSWVRQAPGKGLELVAAINSDGGSTYYP DTMERRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHYDDYYAWFAYWGQGTMVTVS S (SEQ ID NO: 194)and/or a light chain variable region comprising the sequence ofEIVLTQSPATLSLSPGERATLSCRASKSVSTSGYSYMHWYQQKPGQAPRLLIYLASNLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRELPLTFGGGTKVEIK (SEQ ID NO: 195). Insome embodiments, the antibody comprises at least one, two, three, four,five or six hypervariable region (HVR) sequences of antibody Hul 19-122as described in WO 2012/027328. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody Hul 119-122 as described in WO2012/027328.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in WO 2013/028231, which is incorporatedherein by reference in its entirety. In some embodiments, the anti-humanOX40 agonist antibody comprises a heavy chain comprising the sequence ofMYLGLNYVFIVFLLNGVQSEVKLEE SGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIRSKANNHATYYAESVNGRFTISRDDSKS SVYLQMNSLRAEDTGIYYCTWGEVFYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYITCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:196) and/or a lightchain comprising the sequence ofMRPSIQFLGLLLFWLHGAQCDIQMTQSPSSLSASLGGKVTITCKSSQDINKYIAWYQHKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:197). In someembodiments, the anti-human OX40 agonist antibody comprises a heavychain variable region comprising the sequence ofMYLGLNYVFIVFLLNGVQSEVKLEE SGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIRSKANNHATYYAESVNGRFTISRDDSKS SVYLQMNSLRAEDTGIYYCTWGEVFYFDYWGQGTTLTVSS (SEQ ID NO:198) and/or a light chain variable regioncomprising the sequence ofMRPSIQFLGLLLFWLHGAQCDIQMTQSPSSLSASLGGKVTITCKSSQDINKYIAWYQHKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLLTFGAGTKLELK (SEQ IDNO: 199). In some embodiments, the antibody comprises at least one, two,three, four, five, or six hypervariable region (HVR) sequences ofantibody Mab CH 119-43-1 as described in WO 2013/028231. In someembodiments, the antibody comprises a heavy chain variable regionsequence and/or a light chain variable region sequence of antibody MabCH 119-43-1 as described in WO 2013/028231.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in WO 2013/038191, which is incorporatedherein by reference in its entirety. In some embodiments, the anti-humanOX40 agonist antibody comprises a heavy chain variable region comprisingthe sequence ofEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCANYYGSSLSMDYWGQGTSVTVSS (SEQ IDNO:200) and/or a light chain variable region comprising the sequence ofDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKR (SEQ ID NO:201). In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody clone 20E5as described in WO 2013/038191. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody clone 20E5 as described in WO2013/038191.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in WO 2013/038191. In some embodiments, theanti-human OX40 agonist antibody comprises a heavy chain variable regioncomprising the sequence ofEVQLQQSGPELVKPGASVKISCKTSGYTFKDYTMHWVKQSHGKSLEWIGGIYPNNGGSTYNQNFKDKATLTVDKSSSTAYMEFRSLTSEDSAVYYCARMGYHGPHLDFDVWGAGTTVTVSP (SEQ IDNO:202) and/or a light chain variable region comprising the sequence ofDIVMTQSHKFMSTSLGDRVSITCKASQDVGAAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGGGSGTDFTLTISNVQSEDLTDYFCQQYINYPLTFGGGTKLEIKR (SEQ ID NO:203). In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody clone 12H3as described in WO 2013/038191. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody clone 12H3 as described in WO2013/038191.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in WO 2014/148895A1, which is incorporatedherein by reference in its entirety. In some embodiments, the anti-humanOX40 agonist antibody comprises a heavy chain variable region comprisingthe sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWMGYINPYNDGTKYNEKFKGRVTITSDTSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQGTLVTVSS (SEQ IDNO:204) and/or a light chain variable region comprising the sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:205). In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody clone 20E5as described in WO 2014/148895A1. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody clone 20E5 as described in WO2014/148895A1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in WO 2014/148895A1. In some embodiments, theanti-human OX40 agonist antibody comprises a heavy chain variable regioncomprising the sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWMGYINPYNDGTKYNEKFKGRVTITSDTSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQGTLVTVSS (SEQ IDNO:204) and/or a light chain variable region comprising the sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:206). In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody clone 20E5as described in WO 2014/148895A1. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody clone 20E5 as described in WO2014/148895A1.

In some embodiments the OX40 agonist antibody is an anti-human OX40agonist antibody described in WO 2014/148895A1. In some embodiments, theanti-human OX40 agonist antibody comprises a heavy chain variable regioncomprising the sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINPYNDGTKYNEKFKGRATITSDTSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQGTLVTVSS (SEQ IDNO:207) and/or a light chain variable region comprising the sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:205). In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody clone 20E5as described in WO 2014/148895A1. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody clone 20E5 as described in WO2014/148895A1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in WO 2014/148895A1. In some embodiments, theanti-human OX40 agonist antibody comprises a heavy chain variable regioncomprising the sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINPYNDGTKYNEKFKGRATITSDTSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQGTLVTVSS (SEQ IDNO:207) and/or a light chain variable region comprising the sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:206). In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody clone 20E5as described in WO 2014/148895A1. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody clone 20E5 as described in WO2014/148895A1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in WO 2014/148895A1. In some embodiments, theanti-human OX40 agonist antibody comprises a heavy chain variable regioncomprising the sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINPYNDGTKYNEKFKGRATLTSDKSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQGTLVTVSS (SEQ IDNO:208) and/or a light chain variable region comprising the sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:205). In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody clone 20E5as described in WO 2014/148895A1. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody clone 20E5 as described in WO2014/148895A1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in WO 2014/148895A1. In some embodiments, theanti-human OX40 agonist antibody comprises a heavy chain variable regioncomprising the sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINPYNDGTKYNEKFKGRATLTSDKSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQGTLVTVSS (SEQ IDNO:208) and/or a light chain variable region comprising the sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:206). In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody clone 20E5as described in WO 2014/148895A1. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody clone 20E5 as described in WO2014/148895A1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in WO 2014/148895A1. In some embodiments, theanti-human OX40 agonist antibody comprises a heavy chain variable regioncomprising the sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWMGGIYPNNGGSTYNQNFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDVWGQGTTVTV SS (SEQ IDNO:209) and/or a light chain variable region comprising the sequence ofDIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:210). In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody clone 12H3as described in WO 2014/148895A1. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody clone 12H3 as described in WO2014/148895A1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in WO 2014/148895A1. In some embodiments, theanti-human OX40 agonist antibody comprises a heavy chain variable regioncomprising the sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWMGGIYPNNGGSTYNQNFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDVWGQGTTVTV SS (SEQ IDNO:209) and/or a light chain variable region comprising the sequence ofDIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHTGVPDRFSGGGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:211). In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody clone 12H3as described in WO 2014/148895A1. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody clone 12H3 as described in WO2014/148895A1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in WO 2014/148895A1. In some embodiments, theanti-human OX40 agonist antibody comprises a heavy chain variable regioncomprising the sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWIGGIYPNNGGSTYNQNFKDRVTLTADKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDVWGQGTTVTVS S (SEQ IDNO:212) and/or a light chain variable region comprising the sequence ofDIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:210). In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody clone 12H3as described in WO 2014/148895A1. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody clone 12H3 as described in WO2014/148895A1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in WO 2014/148895A1. In some embodiments, theanti-human OX40 agonist antibody comprises a heavy chain variable regioncomprising the sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWIGGIYPNNGGSTYNQNFKDRVTLTADKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDVWGQGTTVTVS S (SEQ IDNO:212) and/or a light chain variable region comprising the sequence ofDIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHTGVPDRFSGGGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:211). In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody clone 12H3as described in WO 2014/148895A1. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody clone 12H3 as described in WO2014/148895A1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in WO 2014/148895A1. In some embodiments, theanti-human OX40 agonist antibody comprises a heavy chain variable regioncomprising the sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWIGGIYPNNGGSTYNQNFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDVWGQGTTVTVS S (SEQ IDNO:213) and/or a light chain variable region comprising the sequence ofDIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:210). In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody clone 12H3as described in WO 2014/148895A1. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody clone 12H3 as described in WO2014/148895A1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40agonist antibody described in WO 2014/148895A1. In some embodiments, theanti-human OX40 agonist antibody comprises a heavy chain variable regioncomprising the sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWIGGIYPNNGGSTYNQNFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDVWGQGTTVTVS S (SEQ IDNO:213) and/or a light chain variable region comprising the sequence ofDIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHTGVPDRFSGGGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:211). In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody clone 12H3as described in WO 2014/148895A1. In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody clone 12H3 as described in WO2014/148895A1.

In some embodiments, the agonist anti-human OX40 antibody is L106 BD(Pharmingen Product #340420). In some embodiments, the antibodycomprises at least one, two, three, four, five or six hypervariableregion (HVR) sequences of antibody L106 (BD Pharmingen Product #340420).In some embodiments, the antibody comprises a heavy chain variableregion sequence and/or a light chain variable region sequence ofantibody L106 (BD Pharmingen Product #340420).

In some embodiments, the agonist anti-human OX40 antibody is ACT35(Santa Cruz Biotechnology, Catalog #20073). In some embodiments, theantibody comprises at least one, two, three, four, five or sixhypervariable region (HVR) sequences of antibody ACT35 (Santa CruzBiotechnology, Catalog #20073). In some embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody ACT35 (Santa Cruz Biotechnology,Catalog #20073).

In some embodiments, the OX40 agonist antibody is MEDI6469. In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody MEDI6469.In some embodiments, the antibody comprises a heavy chain variableregion sequence and/or a light chain variable region sequence ofantibody MEDI6469.

In some embodiments, the OX40 agonist antibody is MEDI0562. In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody MEDI0562.In some embodiments, the antibody comprises a heavy chain variableregion sequence and/or a light chain variable region sequence ofantibody MEDI0562.

In some embodiments, the OX40 agonist antibody is an agonist antibodythat binds to the same epitope as any one of the OX40 agonist antibodiesset forth above.

In some embodiments, the anti-human OX40 agonist antibody has afunctional Fc region. In some embodiments, the Fc region is human IgG1.In some embodiments, the Fc region is human IgG4. In some embodiments,the anti-human OX40 agonist antibody is engineered to increase effectorfunction (e.g., compared to effector function in a wild-type IgG1). Insome embodiments, the antibody has increased binding to a Fc receptor.In some embodiments, the antibody lacks fucose attached (directly orindirectly) to the Fc region. For example, the amount of fucose in suchantibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from20% to 40%. In some embodiments, the Fc region comprises bisectedoligosaccharides, e.g., in which a biantennary oligosaccharide attachedto the Fc region of the antibody is bisected by GlcNAc. In someembodiments, the antibody comprises an Fc region with one or more aminoacid substitutions which improve ADCC, e.g., substitutions at positions298, 333, and/or 334 of the Fc region (EU numbering of residues).

OX40 agonists useful for the methods described herein are in no wayintended to be limited to antibodies. Non-antibody OX40 agonists arecontemplated and well known in the art.

As described above, OX40L (also known as CD134L) serves as a ligand forOX40. As such, agonists that present part or all of OX40L may serve asOX40 agonists. In some embodiments, an OX40 agonist may include one ormore extracellular domains of OX40L. Examples of extracellular domainsof OX40L may include OX40-binding domains. In some embodiments, an OX40agonist may be a soluble form of OX40L that includes one or moreextracellular domains of OX40L but lacks other, insoluble domains of theprotein, e.g., transmembrane domains. In some embodiments, an OX40agonist is a soluble protein that includes one or more extracellulardomains of OX40L able to bind OX40L. In some embodiments, an OX40agonist may be linked to another protein domain, e.g., to increase itseffectiveness, half-life, or other desired characteristics. In someembodiments, an OX40 agonist may include one or more extracellulardomains of OX40L linked to an immunoglobulin Fc domain.

In some embodiments, an OX40 agonist may be any one of the OX40 agonistsdescribed in U.S. Pat. No. 7,696,175.

In some embodiments, an OX40 agonist may be an oligomeric or multimericmolecule. For example, an OX40 agonist may contain one or more domains(e.g., a leucine zipper domain) that allows proteins to oligomerize. Insome embodiments, an OX40 agonist may include one or more extracellulardomains of OX40L linked to one or more leucine zipper domains.

In some embodiments, an OX40 agonist may be any one of the OX40 agonistsdescribed in European Patent No. EP0672141 B1.

In some embodiments, an OX40 agonist may be a trimeric OX40L fusionprotein. For example, an OX40 agonist may include one or moreextracellular domains of OX40L linked to an immunoglobulin Fc domain anda trimerization domain (including without limitation an isoleucinezipper domain).

In some embodiments, an OX40 agonist may be any one of the OX40 agonistsdescribed in International Publication No. WO2006/121810, such as anOX40 immunoadhesin. In some embodiments, the OX40 immunoadhesin may be atrimeric OX40-Fc protein. In some embodiments, the OX40 agonist isMEDI6383.

In a further aspect of the invention, an anti-OX40 antibody according toany of the above embodiments is a monoclonal antibody, including achimeric, humanized or human antibody. In one embodiment, an anti-OX40antibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody,or F(ab′)₂ fragment. In another embodiment, the antibody is a fulllength antibody, e.g., an intact IgG1 antibody or other antibody classor isotype as defined herein. In some embodiments, the antibody is afull length intact IgG4 antibody.

IV. Antibody Preparation

An anti-angiogenesis antibody (e.g., an anti-VEGF antibody) and/or ananti-OX40 antibody according to any of the above embodiments mayincorporate any of the features, singly or in combination, as describedin Sections 1-7 below: 1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from10⁻⁹ M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA). In one embodiment, an RIA is performed with the Fab versionof an antibody of interest and its antigen. For example, solutionbinding affinity of Fabs for antigen is measured by equilibrating Fabwith a minimal concentration of (¹²⁵I)-labeled antigen in the presenceof a titration series of unlabeled antigen, then capturing bound antigenwith an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol.Biol. 293:865-881(1999)). To establish conditions for the assay,MICROTITER® multi-well plates (Thermo Scientific) are coated overnightwith 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mMsodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovineserum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150μl/well of scintillant (MICROSCINT-20™; Packard) is added, and theplates are counted on a TOPCOUNT™ gamma counter (Packard) for tenminutes. Concentrations of each Fab that give less than or equal to 20%of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using a BIACORE® surfaceplasmon resonance assay. For example, an assay using a BIACORE®-2000 ora BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) is performed at 25° C.with immobilized antigen CM5 chips at ˜10 response units (RU). In oneembodiment, carboxymethylated dextran biosensor chips (CM5, BIACORE,Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μl/min. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiokoff/kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). Ifthe on-rate exceeds 106 M-1 s-1 by the surface plasmon resonance assayabove, then the on-rate can be determined by using a fluorescentquenching technique that measures the increase or decrease influorescence emission intensity (excitation=295 nm; emission=340 nm, 16nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) inPBS, pH 7.2, in the presence of increasing concentrations of antigen asmeasured in a spectrometer, such as a stop-flow equipped spectrophometer(Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer(ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthiin, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies arealso described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); USPatent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing specificity determining region(SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimkaet al., Br. J. Cancer, 83:252-260 (2000) (describing the “guidedselection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Nod Acad Sci. USA, 103:3557-3562 (2006).Additional methods include those described, for example, in U.S. Pat.No. 7,189,826 (describing production of monoclonal human IgM antibodiesfrom hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268(2006) (describing human-human hybridomas). Human hybridoma technology(Trioma technology) is also described in Vollmers and Brandlein,Histology and Histopathology, 20(3):927-937 (2005) and Vollmers andBrandlein, Methods and Findings in Experimental and ClinicalPharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. inMethods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for OX40 and the other is for any other antigen. Incertain embodiments, bispecific antibodies may bind to two differentepitopes of OX40. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express OX40. Bispecific antibodies canbe prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBOJ. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S.Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see,e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to OX40 as well asanother, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table A under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table A under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE A Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or residues that contact antigen,with the resulting variant VH or VL being tested for binding affinity.Affinity maturation by constructing and reselecting from secondarylibraries has been described, e.g., in Hoogenboom et al. in Methods inMolecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa,N.J., (2001).) In some embodiments of affinity maturation, diversity isintroduced into the variable genes chosen for maturation by any of avariety of methods (e.g., error-prone PCR, chain shuffling, oroligonucleotide-directed mutagenesis). A secondary library is thencreated. The library is then screened to identify any antibody variantswith the desired affinity. Another method to introduce diversityinvolves HVR-directed approaches, in which several HVR residues (e.g.,4-6 residues at a time) are randomized. HVR residues involved in antigenbinding may be specifically identified, e.g., using alanine scanningmutagenesis or modeling. CDR-H3 and CDR-L3 in particular are oftentargeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout +3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);5,821,337 (see Bruggemann, M. et al., J. Exp. Med 166:1351-1361 (1987)).Alternatively, non-radioactive assays methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the antibody is unable to bind C1qand hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

A. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-OX40 antibody described herein isprovided. Such nucleic acid may encode an amino acid sequence comprisingthe VL and/or an amino acid sequence comprising the VH of the antibody(e.g., the light and/or heavy chains of the antibody). In a furtherembodiment, one or more vectors (e.g., expression vectors) comprisingsuch nucleic acid are provided. In a further embodiment, a host cellcomprising such nucleic acid is provided. In one such embodiment, a hostcell comprises (e.g., has been transformed with): (1) a vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and an amino acid sequence comprising the VH ofthe antibody, or (2) a first vector comprising a nucleic acid thatencodes an amino acid sequence comprising the VL of the antibody and asecond vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., YO, NSO, Sp20 cell). In one embodiment, a method of makingan anti-OX40 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-OX40 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas YO, NSO and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

B. Assays

Anti-OX40 antibodies provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc. OX40 binding may be determined using methods known in the art andexemplary methods are disclosed herein. In one embodiment, binding ismeasured using radioimmunoassay. An exemplary radioimmunassay isexemplified in the Examples. OX40 antibody is iodinated, and competitionreaction mixtures are prepared containing a fixed concentration ofiodinated antibody and decreasing concentrations of serially diluted,unlabeled OZ X40 antibody. Cells expressing OX40 (e.g., BT474 cellsstably transfected with human OX40) are added to the reaction mixture.Following an incubation, cells are washed to separate the free iodinatedOX40 antibody from the OX40 antibody bound to the cells. Level of boundiodinated OX40 antibody is determined, e.g., by counting radioactivityassociated with cells, and binding affinity determined using standardmethods. In another embodiment, ability of OX40 antibody to bind tosurface-expressed OX40 (e.g., on T cell subsets) is assessed using flowcytometry. Peripheral white blood cells are obtained (e.g., from human,cynomolgus monkey, rat or mouse) and cells are blocked with serum.Labeled OX40 antibody is added in serial dilutions, and T cells are alsostained to identify T cell subsets (using methods known in the art).Following incubation of the samples and washing, the cells are sortedusing flow cytometer, and data analyzed using methods well known in theart. In another embodiment, OX40 binding may be analyzed using surfaceplasmon resonance. An exemplary surface plasmon resonance method isexemplified in the Examples.

In another aspect, competition assays may be used to identify anantibody that competes with any of the anti-OX40 antibodies disclosedherein for binding to OX40, or to identify an antibody that competeswith any of the anti-VEGF antibodies disclosed herein for binding toVEGF. In certain embodiments, such a competing antibody binds to thesame epitope (e.g., a linear or a conformational epitope) that is boundby any of the anti-OX40 antibodies disclosed herein. In certainembodiments, such a competing antibody binds to the same epitope (e.g.,a linear or a conformational epitope, or the A4.6.1 epitope) that isbound by any of the anti-VEGF antibodies disclosed herein. Detailedexemplary methods for mapping an epitope to which an antibody binds areprovided in Morris (1996) “Epitope Mapping Protocols,” in Methods inMolecular Biology vol. 66 (Humana Press, Totowa, N.J.). A competitionassay is exemplified in the Examples.

In an exemplary competition assay, immobilized OX40 or VEGF is incubatedin a solution comprising a first labeled antibody that binds to OX40 orVEGF, respectively (e.g., mab 1A7.gr.1 or mab 3C8.gr5 for OX40, orA4.6.1 for VEGF) and a second unlabeled antibody that is being testedfor its ability to compete with the first antibody for binding to OX40or VEGF, respectively. The second antibody may be present in a hybridomasupernatant. As a control, immobilized OX40 or VEGF, respectively, isincubated in a solution comprising the first labeled antibody but notthe second unlabeled antibody. After incubation under conditionspermissive for binding of the first antibody to OX40 or VEGF,respectively, excess unbound antibody is removed, and the amount oflabel associated with immobilized OX40 or VEGF, respectively, ismeasured. If the amount of label associated with immobilized OX40 orVEGF, respectively, is substantially reduced in the test sample relativeto the control sample, then that indicates that the second antibody iscompeting with the first antibody for binding to OX40 or VEGF,respectively. See Harlow and Lane (1988) Antibodies: A Laboratory Manualch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

2. Activity Assays

In one aspect, assays are provided for identifying anti-OX40 antibodiesthereof having biological activity. Biological activity may include,e.g., binding OX40 (e.g., binding human and/or cynomolgus OX40),increasing OX40-mediated signal transduction (e.g., increasingNFkB-mediated transcription), depleting cells that express human OX40(e.g., T cells), depleting cells that express human OX40 by ADCC and/orphagocytosis, enhancing T effector cell function (e.g., CD4+ effector Tcell), e.g., by increasing effector T cell proliferation and/orincreasing cytokine production (e.g., gamma interferon) by effector Tcells, enhancing memory T cell function (e.g., CD4+ memory T cell),e.g., by increasing memory T cell proliferation and/or increasingcytokine production by memory T cells (e.g., gamma interferon),inhibiting regulatory T cell function (e.g., by decreasing Tregsuppression of effector T cell function (e.g., CD4+ effector T cellfunction), binding human effector cells. Antibodies having suchbiological activity in vivo and/or in vitro are also provided.

In certain embodiments, an antibody of the invention is tested for suchbiological activity.

T cell costimulation may be assayed using methods known in the art andexemplary methods are disclosed herein. For example, T cells (e.g.,memory or effector T cells) may be obtained from peripheral white bloodcells (e.g., isolated from human whole blood using Ficoll gradientcentrifugation). Memory T cells (e.g., CD4+ memory T cells) or effectorT cells (e.g. CD4+ Teff cells) may be isolated from PBMC using methodsknown in the art. For example, the Miltenyi CD4+ memory T cell isolationkit or Miltenyi naïve CD4+ T cell isolation kit may be used. Isolated Tcells are cultured in the presence of antigen presenting cells (e.g.,irradiated L cells that express CD32 and CD80), and activated byaddition of anti-CD3 antibody in the presence or absence of OX40 agonistantibody. Effect of agonist OX40 antibody of T cell proliferation may bemeasured using methods well known in the art. For example, the CellTiterGlo kit (Promega) may be used, and results read on a Multilabel Reader(Perkin Elmer). Effect of agonist OX40 antibody on T cell function mayalso be determined by analysis of cytokines produced by the T cell. Inone embodiment, production of interferon gamma by CD4+ T cells isdetermined, e.g., by measurement of interferon gamma in cell culturesupernatant. Methods for measuring interferon gamma are well-known inthe art.

Treg cell function may be assayed using methods known in the art andexemplary methods are disclosed herein. In one example, the ability ofTreg to suppress effector T cell proliferation is assayed. T cells areisolated from human whole blood using methods known in the art (e.g.,isolating memory T cells or naïve T cells). Purified CD4+naïve T cellsare labeled (e.g., with CFSE) and purified Treg cells are labeled with adifferent reagent. Irradiated antigen presenting cells (e.g., L cellsexpressing CD32 and CD80) are co-cultured with the labeled purifiednaïve CD4+ T cells and purified Tregs. The co-cultures are activatedusing anti-CD3 antibody and tested in the presence or absence of agonistOX40 antibody. Following a suitable time (e.g., 6 days of coculture),level of CD4+naïve T cell proliferation is tracked by dye dilution inreduced label staining (e.g., reduced CFSE label staining) using FACSanalysis.

OX40 signaling may be assayed using methods well known in the art andexemplary methods are disclosed herein. In one embodiment, transgeniccells are generated that express human OX40 and a reporter genecomprising the NFkB promoter fused to a reporter gene (e.g., betaluciferase). Addition of OX40 agonist antibody to the cells results inincreased NFkB transcription, which is detected using an assay for thereporter gene.

Phagocytosis may be assayed, e.g., by using monocyte-derivedmacrophages, or U937 cells (a human histiocytic lymphoma cells line withthe morphology and characteristics of mature macrophages). OX40expressing cells are added to the monocyte-derived macrophages or U937cells in the presence or absence of anti-OX40 agonist antibody.Following culturing of the cells for a suitable period of time, thepercentage of phagocytosis is determined by examining percentage ofcells that double stain for markers of 1) the macrophage or U937 celland 2) the OX40 expressing cell, and dividing this by the total numberof cells that show markers of the OX40 expressing cell (e.g., GFP).Analysis may be done by flow cytometry. In another embodiment, analysismay be done by fluorescent microscopy analysis.

ADCC may be assayed, e.g., using methods well known in the art.Exemplary methods are described in the definition section and anexemplary assay is disclosed in the Examples. In some embodiments, levelof OX40 is characterized on an OX40 expressing cell that is used fortesting in an ADCC assay. The cell may be stained with a detectablylabeled anti-OX40 antibody (e.g., PE labeled), then level offluorescence determined using flow cytometry, and results presented asmedian fluorescence intensity (MFI). In another embodiment, ADCC may beanalyzed by CellTiter Glo assay kit and cell viability/cytotoxicity maybe determined by chemioluminescence.

The binding affinities of various antibodies to FcγRIA, FcγRIIA,FcγRIIB, and two allotypes of FcγRIIIA (F158 and V158) may be measuredin ELISA-based ligand-binding assays using the respective recombinantFcγ receptors. Purified human Fcγ receptors are expressed as fusionproteins containing the extracellular domain of the receptor y chainlinked to a Gly/6×His/glutathione S-transferase (GST) polypeptide tag atthe C-terminus. The binding affinities of antibodies to those human Fcγreceptors are assayed as follows. For the low-affinity receptors, i.e.FcγRIIA (CD32A), FcγRIIB (CD32B), and the two allotypes of FcγRIIIA(CD16), F-158 and V-158, antibodies may be tested as multimers bycross-linking with a F(ab′)2 fragment of goat anti-human kappa chain(ICN Biomedical; Irvine, Calif.) at an approximate molar ratio of 1:3antibody:cross-linking F(ab′)₂. Plates are coated with an anti-GSTantibody (Genentech) and blocked with bovine serum albumin (BSA). Afterwashing with phosphate-buffered saline (PBS) containing 0.05% Tween-20with an ELx405™ plate washer (Biotek Instruments; Winooski, Vt.), Fcγreceptors are added to the plate at 25 ng/well and incubated at roomtemperature for 1 hour. After the plates are washed, serial dilutions oftest antibodies are added as multimeric complexes and the plates wereincubated at room temperature for 2 hours. Following plate washing toremove unbound antibodies, the antibodies bound to the Fcγ receptor aredetected with horseradish peroxidase (HRP)-conjugated F(ab′)₂ fragmentof goat anti-human F(ab′)₂ (Jackson ImmunoResearch Laboratories; WestGrove, Pa.) followed by the addition of substrate, tetramethylbenzidine(TMB) (Kirkegaard & Perry Laboratories; Gaithersburg, Md.). The platesare incubated at room temperature for 5-20 minutes, depending on the Fcγreceptors tested, to allow color development. The reaction is terminatedwith 1 M H₃PO₄ and absorbance at 450 nm was measured with a microplatereader (SpectraMax®190, Molecular Devices; Sunnyvale, Calif.).Dose-response binding curves are generated by plotting the meanabsorbance values from the duplicates of antibody dilutions against theconcentrations of the antibody. Values for the effective concentrationof the antibody at which 50% of the maximum response from binding to theFcγ receptor is detected (EC50) were determined after fitting thebinding curve with a four-parameter equation using SoftMax Pro(Molecular Devices).

To select for antibodies which induce cell death, loss of membraneintegrity as indicated by, e.g., propidium iodide (PI), trypan blue or7AAD uptake may be assessed relative to control. A PI uptake assay canbe performed in the absence of complement and immune effector cells.OX40 expressing cells are incubated with medium alone or mediumcontaining of the appropriate monoclonal antibody at e.g., about 10μg/ml. The cells are incubated for a time period (e.g., 1 or 3 days).Following each treatment, cells are washed and aliquoted. In someembodiments, cells are aliquoted into 35 mm strainer-capped 12×75 tubes(lml per tube, 3 tubes per treatment group) for removal of cell clumps.Tubes then receive PI (10 μg/ml). Samples may be analyzed using aFACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (BectonDickinson).

Cells for use in any of the above in vitro assays include cells or celllines that naturally express OX40 or that have been engineered toexpress OX40. Such cells include activated T cells, Treg cells andactivated memory T cells that naturally express OX40. Such cells alsoinclude cell lines that express OX40 and cell lines that do not normallyexpress OX40 but have been transfected with nucleic acid encoding OX40.Exemplary cell lines provided herein for use in any of the above invitro assays include transgenic BT474 cells (a human breast cancer cellline) that express human OX40

It is understood that any of the above assays may be carried out usingan immunoconjugate of the invention in place of or in addition to ananti-OX40 antibody.

It is understood that any of the above assays may be carried out usinganti-OX40 antibody and an additional therapeutic agent.

Assays for identifying anti-VEGF antibodies are known in the art. Forexample, antibody affinities may be determined by a surface plasmonresonance based assay (such as the BIAcore assay as described in PCTApplication Publication No. WO2005/012359); enzyme-linkedimmunoabsorbent assay (ELISA); and competition assays (e.g. RIA's), forexample. In certain embodiments, the anti-VEGF antibody of the inventioncan be used as a therapeutic agent in targeting and interfering withdiseases or conditions wherein the VEGF activity is involved. Also, theantibody may be subjected to other biological activity assays, e.g., inorder to evaluate its effectiveness as a therapeutic. Such assays areknown in the art and depend on the target antigen and intended use forthe antibody. Examples include the HUVEC inhibition assay; tumor cellgrowth inhibition assays (as described in WO 89/06692, for example);antibody-dependent cellular cytotoxicity (ADCC) and complement-mediatedcytotoxicity (CDC) assays (U.S. Pat. No. 5,500,362); and agonisticactivity or hematopoiesis assays (see WO 95/27062).

C. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-OX40antibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med. Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re⁸⁶, Re⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or 1123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

OX40 Agonist Antibody Sequences Name SEQUENCE SEQ ID NO: Human OX40LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPG 1 (lacking theFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAG signal peptide)TQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI HVR-H1- DSYMS 2 1A7.gr.1 1A7.gr.21A7.gr.3 1A7.gr.4 1A7.gr.5 1A7.gr.5′ 1A7.gr.6 1A7.gr.7 1A7.gr.7′1A7.gr.NADS 1A7.gr.NADA 1A7.gr.NGDA 1A7.gr.SGDS 1A7.gr.NGSS 1A7.Ala.11A7.Ala.2 1A7.Ala.3 1A7.Ala.4 1A7.Ala.5 1A7.Ala.6 1A7.Ala.7 1A7.Ala.81A7.Ala.9 1A7.Ala.10 1A7.Ala.11 1A7.Ala.12 1A7.Ala.13 1A7.Ala.141A7.Ala.15 1A7.Ala.16 HVR-H2- DMYPDNGDSSYNQKFRE 3 1A7.gr.1 1A7.gr.21A7.gr.3 1A7.gr.4 1A7.gr.5 1A7.gr.5′ 1A7.gr.6 1A7.gr.7 1A7.gr.7′1A7.gr.DA 1A7.gr.ES 1A7.Ala.1 1A7.Ala.2 1A7.Ala.3 1A7.Ala.4 1A7.Ala.51A7.Ala.6 1A7.Ala.7 1A7.Ala.8 1A7.Ala.9 1A7.Ala.10 1A7.Ala.11 1A7.Ala.121A7.Ala.13 1A7.Ala.14 1A7.Ala.15 1A7.Ala.16 HVR-H3- APRWYFSV 4 1A7.gr.11A7.gr.2 1A7.gr.3 1A7.gr.4 1A7.gr.5 1A7.gr.5′ 1A7.gr.6 1A7.gr.71A7.gr.7′ 1A7.gr.DA 1A7.gr.ES 1A7.gr.NADS 1A7.gr.NADA 1A7.gr.NGDA1A7.gr.SGDS 1A7.gr.NGSS 1A7.gr.DANADA 1A7.Ala.1 1A7.Ala.2 1A7.Ala.31A7.Ala.4 1A7.Ala.5 1A7.Ala.6 1A7.Ala.7 1A7-Ala.15 1A7.Ala.16 HVR-L1-RASQDISNYLN 5 1A7.gr.1 1A7.gr.2 1A7.gr.3 1A7.gr.4 1A7.gr.5 1A7.gr.5′1A7.gr.6 1A7.gr.7 1A7.gr.7′ 1A7.gr.DA 1A7.gr.ES 1A7.gr.NADS 1A7.gr.NADA1A7.gr.NGDA 1A7.gr.SGDS 1A7.gr.NGSS 1A7.gr.DANADA 1A7.Ala.1 1A7.Ala.21A7.Ala.3 1A7.Ala.4 1A7.Ala.5 1A7.Ala.6 1A7.Ala.7 1A7.Ala.8 1A7.Ala.91A7.Ala.10 1A7.Ala.11 1A7.Ala.12 1A7.Ala.13 1A7.Ala.14 1A7.Ala.151A7.Ala.16 HVR-L2- YTSRLRS 6 1A7.gr.1 1A7.gr.2 1A7.gr.3 1A7.gr.41A7.gr.5 1A7.gr.5′ 1A7.gr.6 1A7.gr.7 1A7.gr.7′ 1A7.gr.DA 1A7.gr.ES1A7.gr.NADS 1A7.gr.NADA 1A7.gr.NGDA 1A7.gr.SGDS 1A7.gr.NGSS1A7.gr.DANADA 1A7.Ala.1 1A7.Ala.2 1A7.Ala.3 1A7.Ala.4 1A7.Ala.51A7.Ala.6 1A7.Ala.7 1A7.Ala.8 1A7.Ala.9 1A7.Ala.10 1A7.Ala.11 1A7.Ala.121A7.Ala.13 1A7.Ala.14 1A7.Ala.15 1A7.Ala.16 HVR-L3- QQGHTLPPT 7 1A7.gr.11A7.gr.2 1A7.gr.3 1A7.gr.4 1A7.gr.5 1A7.gr.5′ 1A7.gr.6 1A7.gr.71A7.gr.7′ 1A7.gr.DA 1A7.gr.ES 1A7.gr.NADS 1A7.gr.NADA 1A7.gr.NGDA1A7.gr.SGDS 1A7.gr.NGSS 1A7.gr.DANADA 1A7.Ala.8 1A7.Ala.9 1A7.Ala.101A7.Ala.11 1A7.Ala.12 1A7.Ala.13 1A7.Ala.14 1A7.Ala.15 1A7.Ala.16HVR-H1- DAYMS 8 1A7.gr.DA HVR-H1- ESYMS 9 1A7.gr.ES 1A7.gr.DANADAHVR-H2- DMYPDNADSSYNQKFRE 10 1A7.gr.NADS HVR-H2- DMYPDNADASYNQKFRE 111A7.gr.NADA 1A7.gr.DANADA HVR-H2- DMYPDNGDASYNQKFRE 12 1A7.gr.NGDAHVR-H2- DMYPDSGDSSYNQKFRE 13 1A7.gr.SGDS HVR-H2- DMYPDNGSSSYNQKFRE 141A7.gr.NGSS HVR-H3- APRWYFSA 15 1A7.Ala.8 HVR-H3- APRWYASV 16 1A7.Ala.9HVR-H3- APRWAFSV 17 1A7.Ala.10 HVR-H3- APAWYFSV 18 1A7.Ala.11 HVR-H3-APRWYFAV 19 1A7.Ala.12 HVR-H3- APRAYFSV 20 1A7.Ala.13 HVR-H3- AARWYFSV21 1A7.Ala.14 HVR-L3- QQGHTLPAT 22 1A7.Ala.1 HVR-L3- QQGHTAPPT 231A7.Ala.2 HVR-L3- QQGATLPPT 24 1A7.Ala.3 HVR-L3- QQGHALPPT 25 1A7.Ala.4HVR-L3- QQAHTLPPT 26 1A7.Ala.5 HVR-L3- QQGHTLAPT 27 1A7.Ala.6 HVR-L3-QAGHTLPPT 28 1A7.A1a.7 HVR-H1- NYLIE 29 3C8.gr.1 3C8.gr.2 3C8.gr.33C8.gr.4 3C8.gr.5 3C8.gr.5.SG 3C8.gr.5.EG 3C8.gr.5.QG 3C9.gr.5.DQ3C8.gr.5.DA 3C8.gr.6 3C8.gr.7 3C8.gr.8 3C8.gr.9 3C8.gr.10 3C8.gr.113C8.A.1 3C8.A.2 3C8.A.3 3C8.A.4 3C8.A.5 3C8.A.6 3C8.A.7 3C8.A.8 3C8.A.93C8.A.10 HVR-H2- VINPGSGDTYYSEKFKG 30 3C8.gr.1 3C8.gr.2 3C8.gr.33C8.gr.4 3C8.gr.5 3C8.gr.5.SG 3C8.gr.5.EG 3C8.gr.5.QG 3C8.gr.6 3C8.gr.73C8.gr.8 3C8.gr.9 3C8.gr.10 3C8.gr.11 3C8.A.1 3C8.A.2 3C8.A.3 3C8.A.43C8.A.5 3C8.A.6 3C8.A.7 3C8.A.8 3C8.A.9 3C8.A.10 HVR-H2-VINPGSGDAYYSEKFKG 31 3C8.gr.5.DA HVR-H2- VINPGSGDQYYSEKFKG 323C8.gr.5.DQ HVR-H3- DRLDY 33 3C8.gr.1 3C8.gr.2 3C8.gr.3 3C8.gr.43C8.gr.5 3C8.gr.5.SG 3C8.gr.5.EG 3C8.gr.5.QG 3C8.gr.5.DA 3C8.gr.5.DQ3C8.gr.6 3C8.gr.7 3C8.gr.8 3C8.gr.9 3C8.gr.10 3C8.gr.11 3C8.A.1 3C8.A.23C8.A.3 3C8.A.4 3C8.A.5 3C8.A.6 3C8.A.7 HVR-H3- ARLDY 34 3C8.A.8 HVR-H3-DALDY 35 3C8.A.9 HVR-H3- DRADY 36 3C8.A.10 HVR-L1- HASQDISSYIV 373C8.gr.1 3C8.gr.2 3C8.gr.3 3C8.gr.4 3C8.gr.5 3C8.gr.5.SG 3C8.gr.5.EG3C8.gr.5.QG 3C8.gr.5.DA 3C8.gr.5.DQ 3C8.gr.6 3C8.gr.7 3C8.gr.8 3C8.gr.93C8.gr.10 3C8.gr.11 3C8.A.1 3C8.A.2 3C8.A.3 3C8.A.4 3C8.A.5 3C8.A.63C8.A.7 3C8.A.8 3C8.A.9 3C8.A.10 HVR-L2- HGTNLED 38 3C8.gr.1 3C8.gr.23C8.gr.3 3C8.gr.4 3C8.gr.5 3C8.gr.5.DA 3C8.gr.5.DQ 3C8.gr.6 3C8.gr.73C8.gr.8 3C8.gr.9 3C8.gr.10 3C8.gr.11 3C8.A.1 3C8.A.2 3C8.A.3 3C8.A.43C8.A.5 3C8.A.6 3C8.A.7 3C8.A.8 3C8.A.9 3C8.A.10 HVR-L2- HGTNLES 393C8.gr5.SG HVR-L2- HGTNLEE 40 3C8.gr.5.EG HVR-L2- HGTNLEQ 41 3C8.gr.5.QGHVR-L3 VHYAQFPYT 42 3C8.gr.1 3C8.gr.2 3C8.gr.3 3C8.gr.4 3C8.gr.53C8.gr.5.SG 3C8.gr.5.EG 3C8.gr.5.QG 3C8.gr.5.DA 3C8.gr.5.DQ 3C8.gr.63C8.gr.7 3C8.gr.8 3C8.gr.9 3C8.gr.10 3C8.gr.11 3C8.A.8 3C8.A.9 3C8.A.10HVR-L3- AHYAQFPYT 43 3C8.A.1 HVR-L3- VAYAQFPYT 44 3C8.A.2 HVR-L3-VHAAQFPYT 45 3C8.A.3 HVR-L3- VHYAAFPYT 46 3C8.A.4 HVR-L3- VHYAQAPYT 473C8.A.5 HVR-L3- VHYAQFAYT 48 3C8.A.6 HVR-L3- VHYAQFPAT 49 3C8.A.7HVR-H1- DYGVL 50 1D2.gr.1 1D2.gr.2 1D2.gr.3 HVR-H2- MIWSGGTTDYNAAFIS 511D2.gr.1 1D2.gr.2 1D2.gr.3 HVR-H3- EEMDY 52 1D2.gr.1 1D2.gr.2 1D2.gr.3HVR-L1- RASQDISNFLN 53 1D2.gr.1 1D2.gr.2 1D2.gr.3 HVR-L2- YTSRLHS 541D2.gr.1 1D2.gr.2 1D2.gr.3 HVR-L3- QQGNTLPWT 55 1D2.gr.1 1D2.gr.21D2.gr.3 1A7.gr.1 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 56 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.1DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 57 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.gr.2 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 58 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITVDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.2DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 59 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.gr.3 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 60 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTLTVDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.3DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 61 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.gr.4 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 62 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITVDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.4DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKT 63 V_(L)VKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.gr.5 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 64 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITVDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.5DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKT 65 V_(L)VKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.gr.6 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 66 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITVDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.6DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKT 67 V_(L)VKLLIYYTSRLRSGVPSRFSGSGSGKDYTLTISSLQPEDFATYFC QQGHTLPPTFGQGTKVEIK1A7.gr.7 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 68 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITVDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.7DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKT 69 V_(L)VKLLIYYTSRLRSGVPSRFSGSGSGKDYTLTISSLQPEDFATYFC QQGHTLPPTFGQGTKVEIK1A7.gr.DA EVQLVQSGAEVKKPGASVKVSCKASGYTFTDAYMSWVRQAP 70 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.DADIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 71 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.gr.ES EVQLVQSGAEVKKPGASVKVSCKASGYTFTESYMSWVRQAP 72 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.ESDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 73 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.gr.NADS EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 74 V_(H)GQGLEWIGDMYPDNADSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.NADSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 75 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.gr.NADA EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 76 V_(H)GQGLEWIGDMYPDNADASYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.NADADIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 77 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.gr.NGDA EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 78 V_(H)GQGLEWIGDMYPDNGDASYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.NGDADIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 79 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.gr.SGDS EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 80 V_(H)GQGLEWIGDMYPDSGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.SGDSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 81 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.gr.NGSS EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 82 V_(H)GQGLEWIGDMYPDNGSSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.NGSSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 83 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.gr.DANADA EVQLVQSGAEVKKPGASVKVSCKASGYTFTDAYMSWVRQAP 84 V_(H)GQGLEWIGDMYPDNADASYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.DANADADIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 85 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.Ala.1 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 86 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.Ala.1DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 87 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPATFGQGTKVEIK1A7.Ala.2 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 88 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.Ala.2DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 89 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTAPPTFGQGTKVEIK1A7.Ala.3 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 90 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.Ala.3DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 91 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGATLPPTFGQGTKVEIK1A7.Ala.4 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 92 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.Ala.4DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 93 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHALPPTFGQGTKVEIK1A7.Ala.5 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 94 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.Ala.5DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 95 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQAHTLPPTFGQGTKVEIK1A7.Ala.6 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 96 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.Ala.6DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 97 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLAPTFGQGTKVEIK1A7.Ala.7 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 98 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.Ala.7DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 99 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QAGHTLPPTFGQGTKVEIK1A7.Ala.8 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 100 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSAWGQGTLVTVSS 1A7.Ala.8DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 101 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.Ala.9 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 102 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYASVWGQGTLVTVSS 1A7.Ala.9DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 103 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.Ala.10 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 104 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWAFSVWGQGTLVTVSS 1A7.Ala.10DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 105 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.Ala.11 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 106 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPAWYFSVWGQGTLVTVSS 1A7.Ala.11DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 107 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.Ala.12 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 108 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFAVWGQGTLVTVSS 1A7.Ala.12DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 109 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.Ala.13 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 110 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRAYFSVWGQGTLVTVSS 1A7.Ala.13DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 111 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.Ala.14 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 112 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAARWYFSVWGQGTLVTVSS 1A7.Ala.14DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 113 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.Ala.15 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 114 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCALAPRWYFSVWGQGTLVTVSS 1A7.Ala.15DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 115 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK1A7.Ala.16 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 116 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVAAPRWYFSVWGQGTLVTVSS 1A7.Ala.16DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 117 V_(L)PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK3C8.gr.1 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 118 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTITRDTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.1DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKAP 119 V_(L)KLLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK3C8.gr.2 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 120 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTITADTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.2DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKAP 121 V_(L)KLLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK3C8.gr.3 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 122 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.3DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKAP 123 V_(L)KLLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK3C8.gr.4 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 124 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTITADTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.4DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 125 V_(L)KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK3C8.gr.5 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 126 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.5DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 127 V_(L)KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK3C8.gr.5.SG EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 128 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.5.SGDIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 129 V_(L)KGLIYHGTNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCV HYAQFPYTFGQGTKVEIK3C8.gr.5.EG EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 130 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.5.EGDIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 131 V_(L)KGLIYHGTNLEEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK3C8.gr.5.QG EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 132 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.5.QGDIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 133 V_(L)KGLIYHGTNLEQGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK3C8.gr.6 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 134 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTITADTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.6DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 135 V_(L)KGLIYHGTNLEDGVPSRFSGSGSGADYTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK3C8.gr.7 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 136 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.7DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 137 V_(L)KGLIYHGTNLEDGVPSRFSGSGSGADYTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK3C8.gr.8 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 138 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTRDTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.8DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 139 V_(L)KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK3C8.gr.9 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 140 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTRDTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.9DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSP 141 V_(L)KLLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK3C8.gr.10 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 142 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTRDTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.10DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKAF 143 V_(L)KLLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK3C8.gr.11 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 144 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTRDTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.11DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKAP 145 V_(L)KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK 3C8.A.1EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 146 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.A.1DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 147 V_(L)KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC AHYAQFPYTFGQGTKVEIK 3C8.A.2EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 148 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.A.2DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 149 V_(L)KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VAYAQFPYTFGQGTKVEIK 3C8.A.3EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 150 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.A.3DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 151 V_(L)KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHAAQFPYTFGQGTKVEIK 3C8.A.4EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 152 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.A.4DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 153 V_(L)KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAAFPYTFGQGTKVEIK 3C8.A.5EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 154 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.A.5DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 155 V_(L)KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQAPYTFGQGTKVEIK 3C8.A.6EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 156 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.A.6DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 157 V_(L)KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFAYTFGQGTKVEIK 3C8.A.7EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 158 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSLRSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.A.7DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 159 V_(L)KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPATFGQGTKVEIK 3C8.A.8EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 160 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSLRSEDTAVYYCARARLDYWGQGTLVTVSS 3C8.A.8DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 161 V_(L)KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK 3C8.A.9EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 162 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSLRSEDTAVYYCARDALDYWGQGTLVTVSS 3C8.A.9DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 163 V_(L)KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK3C8.A.10 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 164 V_(H)QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSLRSEDTAVYYCARDRADYWGQGTLVTVSS 3C8.A.10DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 165 V_(L)KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK1D2.gr.1 EVQLVESGPGLVKPSETLSLTCTVSGFSLTDYGVLWIRQPPGKG 166 V_(H)LEWIGMIWSGGTTDYNAAFISRVTISVDTSKNQFSLKLSSVTAA DTAVYYCVREEMDYWGQGTLVTVSS1D2.gr.1 DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGKA 167 V_(L)PKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGNTLPWTFGQGTKVEIK1D2.gr.2 EVQLVESGPGLVKPSETLSLTCTVSGFSLTDYGVLWIRQPPGKG 168 V_(H)LEWIGMIWSGGTTDYNAAFISRVTISKDTSKNQVSLKLSSVTA ADTAVYYCVREEMDYWGQGTLVTVSS1D2.gr.2 DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGKA 169 V_(L)PKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGNTLPWTFGQGTKVEIK1D2.gr.3 EVQLVESGPGLVKPSETLSLTCTVSGFSLTDYGVLWVRQPPGK 170 V_(H)GLEWLGMIWSGGTTDYNAAFISRLTISKDTSKNQVSLKLSSVT AADTAVYYCVREEMDYWGQGTLVTVSS1D2.gr.3 DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGKA 171 V_(L)PKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGNTLPWTFGQGTKVEIKCON1 (1A7) X₁X₂YMS, wherein X₁ is D or E, and 172 HVR-H1 X₂ is S or ACON1 (1A7) DMYPDX₁X₂X₃X₄SYNQKFRE, wherein X₁ is N or S,  173 HVR-H2X₁ is A or G, X₃ is D or S, and X₄ is A or S CON1 (1A7)APRWX₁X₂X₃X₄, wherein X₁ is Y or A,  174 HVR-H3X₂ is A or F, X₃ is S or A, and  X₄ is A or V. CON1 (1A7)QX₁X₂X₃X₄X₅X₆X₇T, wherein X₁ is A or Q,  175 HVR-L3X₂ is A or G, X₃ is A or H, X₄ is A or T, X₅ is A or L, X₆ is A or P, and X₇ is A or P. CON2 (3C8)VINPGSGDX₁YYSEKFKG,wherein X₁ is T, A or Q. 176 HVR-H2 CON2 (3C8)HGTNLEX₁,wherein X₁ is S, E, or Q. 177 HVR-L2 CON2 (3C8)X₁X₂YAQFPYX₃, wherein X₁ is V or A, 178 HVR-L3X₂ is H or A, and X₃ is Y or A. 1A7 V_(L)DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGT  179VKLLIYYTSRLRSGVPSRFSGSGSGKDYFLTISNLEQEDVAAYF CQQGHTLPPTFGGGTKLEIK1A7 V_(H) EVQLQQSGPELVKPGASVKISCKASGYTFTDSYMSWVKQSHG  180KTLEWIGDMYPDNGDSSYNQKFREKVTLTVDKSSTTAYMEFRSLTSEDSAVYYCVLAPRWYFSVWGTGTTVTVSS 3C8 V_(L)DILMTQSPSSMSVSLGDTVSITCHASQDISSYIVWLQQKPGKSF  181RGLIYHGTNLEDGIPSRFSGSGSGADYSLTISSLESEDFADYYCV HYAQFPYTFGGGTKLEIK3C8 V_(H) QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPG  182QGLEWIGVINPGSGDTYYSEKFKGKVTLTADKSSSTAYMQLSSLTSEDSAVYFCARDRLDYWGQGTTLTVSS 1A7.gr.5′EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 233 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTLTVDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.7′EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 234 V_(H)GQGLEWIGDMYPDNGDSSYNQKFRERVTLTVDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS

IV. Methods of Treatment

Certain aspects of the present disclosure relate to method for treatingor delaying progression of cancer in an individual comprisingadministering to the individual an effective amount of ananti-angiogenesis agent described herein and an OX40 binding agonistdescribed herein. For example, any of the anti-angiogenesis agents(e.g., anti-VEGF antibodies) and OX40 binding agonists (e.g., anti-humanOX40 antibodies) provided herein may be used in therapeutic methods. Insome embodiments, the individual has cancer or has been diagnosed withcancer. In some embodiments, the treatment results in a sustainedresponse in the individual after cessation of the treatment. In someembodiments, the individual is a human.

In some embodiments, the OX40 binding agonist is administered before theanti-angiogenesis agent, simultaneous with the anti-angiogenesis agent,or after the anti-angiogenesis agent.

Examples of various cancer types that can be treated with ananti-angiogenesis agent (e.g, a VEGF antagonist such as an anti-VEGFantibody like bevacizumab) and an OX40 binding agonist are describedabove. Preferred cancer types include gynecologic cancers (e.g.,ovarian, peritoneal, fallopian tube, cervical, endometrial, vaginal, andvulvar cancer). Additional cancers include epithelial ovarian cancer,fallopian tube cancer, primary peritoneal cancer, squamous cell cancer,lung cancer (including small-cell lung cancer, non-small cell lungcancer, adenocarcinoma of the lung, and squamous carcinoma of the lung),cancer of the peritoneun, hepatocellular cancer, gastric or stomachcancer (including gastrointestinal cancer), pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer (including platinumsensitive and platinum resistant ovarian cancer), liver cancer, bladdercancer, hepatoma, neuroblastoma, melanoma, breast cancer, colon cancer,colorectal cancer, fallopian tube, peritoneal, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, soft-tissuesarcoma, kaposi's sarcoma, carcinoid carcinoma, mesothelioma, multiplemyeloma, hepatic carcinoma and various types of head and neck cancer, aswell as B-cell lyrnphoma (including low grade/follicular non-Hodgkin'slymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstromn's Macroglobulinemnia); chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome. In variousembodiments, the cancer that is treated is advanced, refractory,recurrent, chemotherapy-resistant, and/or platinum-resistant cancer.

In some embodiments, examples of cancer further include, but are notlimited to, B-cell lymphoma (including low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), B-cell proliferative disorders, andMeigs' syndrome. More specific examples include, but are not limited to,relapsed or refractory NHL, front line low grade NHL, Stage III/IV NHL,chemotherapy resistant NHL, precursor B lymphoblastic leukemia and/orlymphoma, small lymphocytic lymphoma, B-cell chronic lymphocyticleukemia and/or prolymphocytic leukemia and/or small lymphocyticlymphoma, B-cell prolymphocytic lymphoma, immunocytoma and/orlymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zoneB-cell lymphoma, splenic marginal zone lymphoma, extranodal marginalzone-MALT lymphoma, nodal marginal zone lymphoma, hairy cell leukemia,plasmacytoma and/or plasma cell myeloma, low grade/follicular lymphoma,intermediate grade/follicular NHL, mantle cell lymphoma, follicle centerlymphoma (follicular), intermediate grade diffuse NHL, diffuse largeB-cell lymphoma, aggressive NHL (including aggressive front-line NHL andaggressive relapsed NHL), NHL relapsing after or refractory toautologous stem cell transplantation, primary mediastinal large B-celllymphoma, primary effusion lymphoma, high grade immunoblastic NHL, highgrade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulkydisease NHL, Burkitt's lymphoma, precursor (peripheral) large granularlymphocytic leukemia, mycosis fungoides and/or Sezary syndrome, skin(cutaneous) lymphomas, anaplastic large cell lymphoma, angiocentriclymphoma.

In some embodiments, examples of cancer further include, but are notlimited to, B-cell proliferative disorders, which further include, butare not limited to, lymphomas (e.g., B-Cell Non-Hodgkin's lymphomas(NHL)) and lymphocytic leukemias. Such lymphomas and lymphocyticleukemias include e.g. a) follicular lymphomas, b) Small Non-CleavedCell Lymphomas/Burkitt's lymphoma (including endemic Burkitt's lymphoma,sporadic Burkitt's lymphoma and Non-Burkitt's lymphoma), c) marginalzone lymphomas (including extranodal marginal zone B-cell lymphoma(Mucosa-associated lymphatic tissue lymphomas, MALT), nodal marginalzone B-cell lymphoma and splenic marginal zone lymphoma), d) Mantle celllymphoma (MCL), e) Large Cell Lymphoma (including B-cell diffuse largecell lymphoma (DLCL), Diffuse Mixed Cell Lymphoma, ImmunoblasticLymphoma, Primary Mediastinal B-Cell Lymphoma, AngiocentricLymphoma-Pulmonary B-Cell Lymphoma), f) hairy cell leukemia, g)lymphocytic lymphoma, Waldenstrom's macroglobulinemia, h) acutelymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL)/smalllymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, i) plasmacell neoplasms, plasma cell myeloma, multiple myeloma, plasmacytoma,and/or j) Hodgkin's disease.

In some embodiments of any of the methods, the cancer is a B-cellproliferative disorder. In some embodiments, the B-cell proliferativedisorder is lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL,relapsed aggressive NHL, relapsed indolent NHL, refractory NHL,refractory indolent NHL, chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acutelymphocytic leukemia (ALL), or mantle cell lymphoma. In someembodiments, the B-cell proliferative disorder is NHL, such as indolentNHL and/or aggressive NHL. In some embodiments, the B-cell proliferativedisorder is indolent follicular lymphoma or diffuse large B-celllymphoma.

In some embodiments, the anti-angiogenesis agent and/or the OX40 bindingagonist are administered intravenously, intramuscularly, subcutaneously,intracerobrospinally, topically, orally, transdermally,intraperitoneally, intraorbitally, by implantation, by inhalation,intrathecally, intraventricularly, intra-articularly, intrasynovially,or intranasally. The VEOF antagonist and/or the OX40 binding agonist(e.g., an anti-VEGF antibody, such as bevacizumab), optionally incombination with one or more chemotherapeutic agents (e.g., carboplatinand/or paclitaxel), are administered to a human patient in accordancewith known methods, such as intravenous administration, e.g., as a bolusor by continuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes.Intravenous administration of the antibody is preferred.

Pharmaceutical formulations of an anti-OX40 antibody and/oranti-angiogenesis agent as described herein are prepared by mixing suchantibody or other agent having the desired degree of purity with one ormore optional pharmaceutically acceptable carriers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable carriers are generally nontoxic to recipients at the dosagesand concentrations employed, and include, but are not limited to:buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

In some embodiments, a “histidine buffer” is a buffer comprisinghistidine ions. Examples of histidine buffers include histidinechloride, histidine acetate, histidine phosphate, histidine sulfate. Thepreferred histidine buffer identified in the examples herein was foundto be histidine acetate. In the preferred embodiment, the histidineacetate buffer is prepared by titrating L-histidine (free base, solid)with acetic acid (liquid). In some embodiments, the histidine buffer orhistidine-acetate buffer is at pH 5.0 to 6.0, in some embodiments, pH5.3 to 5.8.

In some embodiments, a “saccharide” herein comprises the generalcomposition (CH2O)n and derivatives thereof, including monosaccharides,disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducingsugars, nonreducing sugars, etc. Examples of saccharides herein includeglucose, sucrose, trehalose, lactose, fructose, maltose, dextran,glycerin, dextran, erythritol, glycerol, arabitol, sylitol, sorbitol,mannitol, mellibiose, melezitose, raffinose, mannotriose, stachyose,maltose, lactulose, maltulose, glucitol, maltitol, lactitol,iso-maltulose, etc. In some embodiments, the saccharide is a nonreducingdisaccharide, such as trehalose or sucrose.

In some embodiments herein, a “surfactant” refers to a surface-activeagent, preferably a nonionic surfactant. Examples of surfactants hereininclude polysorbate (for example, polysorbate 20 and polysorbate 80);poloxamer (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS);sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-,linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.); polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g. Pluronics, PF68 etc); etc. In someembodiments, the surfactant is polysorbate 20. In some embodiments, thesurfactant is polysorbate 80.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, it may be desirable to further provide an additionalmedicament (examples of which are provided herein). Such activeingredients are suitably present in combination in amounts that areeffective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

In some embodiments, provided herein are pharmaceutical formulationscomprising: (a) any of the anti-human OX40 agonist antibodies describedherein; (b) a histidine buffer at pH 5.0-6.0.

In some embodiments, provided herein are pharmaceutical formulationscomprising: (a) any of the anti-human OX40 agonist antibodies describedherein; (b) a histidine buffer at pH 5.0-6.0; (c) a saccharide; and (d)a surfactant.

In some embodiments of any of the formulations, the anti-human OX40agonist antibody is present at a concentration between about 10 mg/mLand about 100 mg/mL (e.g. about 15 mg/mL, 18 mg/mL, 20 mg/mL, 60 mg/mL,and 75 mg/mL). In some embodiments, the anti-human OX40 agonist antibodyis present at a concentration of about 20 mg/mL. In some embodiments,the anti-human OX40 agonist antibody is present at a concentration ofabout 50 mg/mL. In some embodiments, the anti-human OX40 agonistantibody is present at a concentration of about 60 mg/mL. In someembodiments, the anti-human OX40 agonist antibody is present at aconcentration of about 70 mg/mL.

In some embodiments of any of the formulations, the saccharide ispresent at a concentration of about 75 mM to about 360 mM (e.g., about100 mM, about 120 mM, about 240 mM, about 320 mM to about 360 mM). Insome embodiments, the saccharide is present at a concentration of about120 mM. In some embodiments, the saccharide is present at aconcentration of about 240 mM. In some embodiments, the saccharide ispresent at a concentration of about 320 mM. In some embodiments, thesaccharide is a disaccharide. In some embodiments, the disaccharide istrehalose. In some embodiments, the disaccharide is sucrose.

In some embodiments of any of the formulations, the histidine buffer isat a concentration of about 1 mM to about 50 mM (e.g. about 1 mM toabout 25 mM). In some embodiments, the histidine buffer is at aconcentration of about 10 mM. In some embodiments, the histidine bufferis at a concentration of about 20 mM. In some embodiments, the histidinebuffer is at a concentration of about 30 mM. In some embodiments, thehistidine buffer is histidine acetate.

In some embodiments of any of the formulations, the surfactant ispolysorbate (e.g., polysorbate 20 or polysorbate 40), poloxamer (e.g.poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurelsulfate; or sodium octyl glycoside.

In some embodiments of any of the formulations, the surfactant ispolysorbate. In some embodiments, the polysorbate is present at aconcentration of about 0.005% to about 0.1%. In some embodiments, thepolysorbate is present at a concentration of about 0.005%. In someembodiments, the polysorbate is present at a concentration of about0.02%. In some embodiments, the polysorbate is present at aconcentration of about 0.04%. In some embodiments, the polysorbate ispresent at a concentration of about 0.06%. In some embodiments, thepolysorbate is polysorbate 20. In some embodiments, the polysorbate ispolysorbate 80.

In some embodiments of any of the formulations, the formulation isdiluted with a diluent (e.g., 0.9% NaCl). In some embodiments, theanti-human OX40 agonist antibody is present at a concentration of about1 mg/mL.

In particular, provided herein are pharmaceutical formulationscomprising (a) any of the anti-human OX40 agonist antibodies describedherein, (b) a polysorbate, wherein the polysorbate concentration isabout 0.005% to about 0.1%.; and (c) a histidine buffer (e.g., ahistidine buffer at a pH between 5.0 and 6.0).

In some embodiments, the pharmaceutical formulation comprises (a) any ofthe anti-human OX40 agonist antibodies described herein, (b) apolysorbate, wherein the polysorbate concentration is about 0.02% toabout 0.06%; (c) a histidine buffer (e.g., a histidine buffer at a pHbetween 5.0 and 6.0); and a saccharide, wherein the saccharideconcentration is about 120 mM to about 320 mM. In some embodiments, thesaccharide is sucrose.

In some embodiments, the pharmaceutical formulation comprises (a) any ofthe anti-human OX40 agonist antibodies described herein, (b) apolysorbate, wherein the polysorbate concentration is about 0.02% toabout 0.06%, wherein the polysorbate is polysorbate 20 or polysorbate40; (c) a histidine acetate buffer (e.g., a histidine acetate buffer ata pH between 5.0 and 6.0); and a saccharide (e.g., sucrose) at aconcentration of about 120 mM to about 320 mM.

In some embodiments, the pharmaceutical formulation comprises (a) any ofthe anti-human OX40 agonist antibodies described herein, (b) polysorbate20, wherein the polysorbate concentration is about 0.02% to about 0.06%;(c) a histidine acetate buffer (e.g., a histidine acetate buffer at a pHbetween 5.0 and 6.0); and (d) sucrose, wherein the sucrose concentrationis about 120 mM to about 320 mM.

In some embodiments, the pharmaceutical formulation comprises (a) any ofthe anti-human OX40 agonist antibodies described herein, (b) polysorbate40, wherein the polysorbate concentration is about 0.02% to about 0.06%;(c) a histidine acetate buffer (e.g., a histidine acetate buffer at a pHbetween 5.0 and 6.0); and sucrose, wherein the sucrose concentration isabout 120 mM to about 320 mM.

In some embodiments, the pharmaceutical formulation comprises (a) any ofthe anti-human OX40 agonist antibodies described herein, (b) polysorbate20, wherein the polysorbate concentration is about 0.02%; (c) ahistidine acetate buffer at pH 6.0; and (d) sucrose, wherein the sucroseconcentration is about 320 mM.

In some embodiments, the pharmaceutical formulation comprises (a) any ofthe anti-human OX40 agonist antibodies described herein, (b) polysorbate20, wherein the polysorbate concentration is about 0.02%; (c) ahistidine acetate buffer at pH 5.5; and (d) sucrose, wherein the sucroseconcentration is about 240 mM.

In some embodiments, the pharmaceutical formulation comprises (a) any ofthe anti-human OX40 agonist antibodies described herein, (b) polysorbate20, wherein the polysorbate concentration is about 0.04%; (c) ahistidine acetate buffer at pH 6.0; and (d) sucrose, wherein the sucroseconcentration is about 120 mM.

In some embodiments, the pharmaceutical formulation comprises (a) any ofthe anti-human OX40 agonist antibodies described herein, (b) polysorbate40, wherein the polysorbate concentration is about 0.04%; (c) ahistidine acetate buffer at pH 5.0; and (d) sucrose, wherein the sucroseconcentration is about 240 mM.

In some embodiments, the pharmaceutical formulation comprises (a) any ofthe anti-human OX40 agonist antibodies described herein, (b) polysorbate40, wherein the polysorbate concentration is about 0.04%; (c) ahistidine acetate buffer at pH 6.0; and (d) sucrose, wherein the sucroseconcentration is about 120 mM.

In some embodiments, the pharmaceutical formulation is a liquidpharmaceutical formulation. In some embodiments, the pharmaceuticalformulation is a stable pharmaceutical formulation. In some embodiments,the pharmaceutical formulation is a stable liquid pharmaceuticalformulation.

In some embodiments of any of the pharmaceutical formulations describedherein, the anti-human OX40 agonist antibody of the pharmaceuticalformulation is present at a concentration between about 10 mg/mL andabout 100 mg/mL. In some embodiments, the concentration of the humanOX40 agonist antibody is between about any of 10 mg/mL to 50 mg/mL, 10mg/mL to 75 mg/mL, 25 mg/mL to 75 mg/mL, 50 mg/mL to 100 mg/mL, 50 mg/mLto 75 mg/mL, and/or 75 mg/mL to 100 mg/mL. In some embodiments, theconcentration of the human OX40 agonist antibody is greater than aboutany of 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, or100 mg/mL.

The pharmaceutical formulation preferably comprises a polysorbate. Thepolysorbate is generally included in an amount which reduces aggregateformation (such as that which occurs upon shaking or shipping). Examplesof polysorbate include, but are not limited to, polysorbate 20(polyoxyethylene (20) sorbitan monolaurate), polysorbate 40(polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60(polyoxyethylene (20) sorbitan monostearate), and/or polysorbate 80(polyoxyethylene (20) sorbitan monooleate). In some embodiments, thepolysorbate is polysorbate 20 (polyoxyethylene (20) sorbitanmonolaurate). In some embodiments of any of the pharmaceuticalformulations described herein, the polysorbate concentration issufficient to minimize aggregation and/or maintain stability upon longterm storage and/or during administration (e.g., after dilution in an IVbag). In some embodiments, the polysorbate concentration is about 0.005%w/v, about 0.02% w/v, about 0.04% w/v and less than about 0.1% w/v. Insome embodiments, the polysorbate concentration is greater than 0.01%w/v and less than about 0.1% w/v. In some embodiments, the polysorbateconcentration is about any of 0.005% w/v, about 0.02% w/v, 0.03% w/v,0.04% w/v, or 0.05% w/v. In some embodiments, the polysorbate is presentat a concentration of about 0.04% w/v. In some embodiments, thepolysorbate is present at a concentration of about 0.02% w/v.

The pharmaceutical formulation preferably comprises a saccharide.Saccharides include monosaccharides, disaccharides, trisaccharides,polysaccharides, sugar alcohols, reducing sugars, nonreducing sugars,etc. Further examples of saccharides include, but are not limited to,glucose, sucrose, trehalose, lactose, fructose, maltose, dextran,glycerin, dextran, erythritol, glycerol, arabitol, sylitol, sorbitol,mannitol, mellibiose, melezitose, raffinose, mannotriose, stachyose,maltose, lactulose, maltulose, glucitol, maltitol, lactitol,iso-maltulose, etc. In some embodiments, the saccharide is adisaccharide. In some embodiments, the saccharide is a nonreducingdisaccharide. In some embodiments, the saccharide is trehalose.

The saccharide is generally included in an amount which reducesaggregate formation. In some embodiments of any of the pharmaceuticalformulations described herein, the saccharide is present at aconcentration of between about any of 50 mM to 250 mM, 75 mM to 200 mM,75 mM to 150 mM, 100 mM to 150 mM, or 110 mM to 130 mM, or 100 mM to 320mM, or 240 mM to 320 mM, or 240 mM to 400 mM. In some embodiments, thesaccharide is present at a concentration greater than about any of 50mM, 75 mM, 100 mM, 110 mM, or 115 mM. In some embodiments, thesaccharide is present at a concentration of about any of 100 mM, 110 mM,120 mM, 130 mM, or 140 mM. In some embodiments, the saccharide ispresent at a concentration of about 120 mM. In some embodiments of anyof the formulations, the saccharide is present at a concentration ofabout 75 mM to about 360 mM (e.g., about 100 mM, about 120 mM, about 240mM, about 320 mM to about 360 mM). In some embodiments, the saccharideis present at a concentration of about 240 mM. In some embodiments, thesaccharide is present at a concentration of about 320 mM.

The pharmaceutical formulation preferably comprises a histidine buffer.Examples of histidine buffers include, but are not limited to, histidinechloride, histidine succinate, histidine acetate, histidine phosphate,histidine sulfate. In some embodiments, the histidine buffer ishistidine acetate. In some embodiments of any of the pharmaceuticalformulations described herein, the histidine buffer concentration isbetween about any of 1 mM to 50 mM, 1 mM to 35 mM, 1 mM to 25 mM, 1 mMto 20 mM, 7.5 mM to 12.5 mM, or 5 mM to 15 mM, 20 mM to 30 mM, 25 mM to35 mM. In some embodiments, the histidine buffer concentration is aboutany of 5 mM, 7.5 mM, 10 mM, 12.5 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mMor 40 mM. In some embodiments, the histidine buffer concentration isabout 10 mM. In some embodiments, the histidine buffer concentration isabout 20 mM. In some embodiments, the histidine buffer concentration isabout 30 mM. In some embodiments, the histidine buffer concentration isabout 40 mM. In some embodiments of any of the pharmaceuticalformulations described herein, the histidine buffer is at a pH ofbetween pH 5.0 and 6.0, for example, about any of pH 5.0, pH 5.1, pH5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9 or pH 6.0.In some embodiments, the pH is between pH 4.9 to pH 6.3.

The pharmaceutical formulation herein may also contain more than oneactive compound as necessary for the particular indication beingtreated, preferably those with complementary activities that do notadversely affect each other. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

Further, provided herein are vials and methods of filing a vialcomprising a pharmaceutical formulation described herein. In someembodiments, the pharmaceutical formulation is provided inside a vialwith a stopper pierceable by a syringe, preferably in aqueous form. Thevial is desirably stored at about 2-8° C. as well as up to 30° C. for 24hours until it is administered to a subject in need thereof. The vialmay for example be a 15 cc vial (for example for a 200 mg dose).

The pharmaceutical formulation for administration is preferably a liquidformulation (not lyophilized) and has not been subjected to priorlyophilization. While the pharmaceutical formulation may be lyophilized,preferably it is not. In some embodiments of any of the pharmaceuticalformulations, the pharmaceutical formulation, the pharmaceuticalformulation is a lyophilized pharmaceutical formulation. In someembodiments, the pharmaceutical formulation is a liquid formulation. Insome embodiments, the pharmaceutical formulation does not contain atonicifying amount of a salt such as sodium chloride. In someembodiments of any of the pharmaceutical formulations, thepharmaceutical formulation is diluted.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, it may be desirable to further provide an additionalmedicament (examples of which are provided herein). Such activeingredients are suitably present in combination in amounts that areeffective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

In one aspect, an OX40 binding agonist (e.g., an anti-human OX40 agonistantibody) and/or anti-angiogenesis agent (e.g., an anti-VEGF antibody)for use as a medicament is provided. In some embodiments, ananti-angiogenesis agent (e.g., an anti-VEGF antibody) for use as amedicament is provided for treating or delaying progression of cancer inan individual, where the medicament comprises the anti-angiogenesisagent and an optional pharmaceutically acceptable carrier, and where thetreatment comprises administration of the medicament in combination witha composition comprising an OX40 binding agonist and an optionalpharmaceutically acceptable carrier. In other embodiments, an OX40binding agonist (e.g., an anti-human OX40 agonist antibody) for use as amedicament is provided for treating or delaying progression of cancer inan individual, where the medicament comprises the anti-angiogenesisagent and an optional pharmaceutically acceptable carrier, and where thetreatment comprises administration of the medicament in combination witha composition comprising an anti-angiogenesis agent and an optionalpharmaceutically acceptable carrier. In some embodiments, the methodfurther comprises administering to the individual an effective amount ofat least one additional therapeutic agent, e.g., as described below.

In one aspect that may be combined with an anti-angiogenesis agent asdescribed herein, provided is an anti-human OX40 agonist antibody foruse in enhancing immune function (e.g., by upregulating cell-mediatedimmune responses) in an individual having cancer comprisingadministering to the individual an effective amount of the anti-humanagonist OX40 antibody. In one aspect, provided is an anti-human OX40agonist antibody for use in enhancing T cell function in an individualhaving cancer comprising administering to the individual an effectiveamount of the anti-human agonist OX40 antibody. In one aspect, providedare an anti-human OX40 agonist antibody for use in depleting humanOX40-expressing cells (e.g., OX40 expressing T cells, e.g., OX40expressing Treg) comprising administering to the individual an effectiveamount of the anti-human agonist OX40 antibody. In some embodiments,depletion is by ADCC. In some embodiments, depletion is by phagocytosis.Provided is an anti-human OX40 agonist antibody for treating anindividual having tumor immunity.

In a further aspect, the invention provides for the use of an anti-OX40antibody in the manufacture or preparation of a medicament. In a furtheraspect, the invention provides for the use of an anti-VEGF antibody inthe manufacture or preparation of a medicament. In one embodiment, themedicament is for treatment of cancer. In a further embodiment, themedicament is for use in a method of treating cancer comprisingadministering to an individual having cancer an effective amount of themedicament. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, e.g., as described below.

In one aspect, the medicament is for use in enhancing immune function(e.g., by upregulating cell-mediated immune responses) in an individualhaving cancer comprising administering to the individual an effectiveamount of the medicament. In one aspect, the medicament is for use inenhancing T cell function in an individual having cancer comprisingadministering to the individual an effective amount of the medicament.In some embodiments, the T cell dysfunctional disorder is cancer. In oneaspect, the medicament is for use in depleting human OX40-expressingcells (e.g., cell expressing high OX40, e.g., OX40 expressing T cells)comprising administering to the individual an effective amount of themedicament. In some embodiments, depletion is by ADCC. In someembodiments, depletion is by phagocytosis. In one aspect, the medicamentis for treating an individual having tumor immunity.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the OX40 binding agonists (e.g., anti-OX40 antibodies)and/or anti-angiogenesis agents (e.g., anti-VEGF antibodies) providedherein, e.g., for use in any of the above therapeutic methods. In oneembodiment, a pharmaceutical formulation comprises any of the anti-OX40antibodies and/or anti-angiogenesis agents (e.g., anti-VEGF antibodies)provided herein and a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical formulation comprises any of the anti-OX40antibodies provided herein and/or anti-angiogenesis agents (e.g.,anti-VEGF antibodies) and at least one additional therapeutic agent,e.g., as described below.

In some embodiments of any of the methods of the invention, theanti-human OX40 agonist antibodies inhibits tumor immunity by inhibitingTreg function (e.g., inhibiting the suppressive function of Tregs),killing OX40 expressing cells (e.g., cells that express high levels ofOX40), increasing effector T cell function and/or increasing memory Tcell function. In some embodiments of any of the methods of theinvention, the anti-human OX40 agonist antibodies treat cancer byinhibiting Treg function (e.g., inhibiting the suppressive function ofTregs), killing OX40 expressing cells (e.g., cells that express highlevels of OX40), increasing effector T cell function and/or increasingmemory T cell function. In some embodiments of any of the methods of theinvention, the anti-human OX40 agonist antibodies enhance immunefunction by inhibiting Treg function (e.g., inhibiting the suppressivefunction of Tregs), killing OX40 expressing cells (e.g., cells thatexpress high levels of OX40), increasing effector T cell function and/orincreasing memory T cell function. In some embodiments of any of themethods of the invention, the anti-human OX40 agonist antibodies enhanceT cell function by inhibiting Treg function (e.g., inhibiting thesuppressive function of Tregs), killing OX40 expressing cells (e.g.,cells that express high levels of OX40), increasing effector T cellfunction and/or increasing memory T cell function.

In some embodiments of any of the methods, the anti-human OX40 agonistantibody is a depleting anti-human agonist antibody. In someembodiments, treatment with the anti-human OX40 agonist antibody resultsin cell depletion (e.g., depletion of OX40-expressing cells, e.g.,depletion of cells that express high levels of OX40). In someembodiments, depletion is by ADCC. In some embodiments, depletion is byphagocytosis.

In some embodiments of any of the methods, the anti-human OX40 agonistantibody inhibits Treg function, e.g., by inhibiting Treg suppression ofeffector and/or memory T cell function (in some embodiments, effector Tcell and/or memory T cell proliferation and/or cytokine secretion),relative to Treg function prior to administration of the OX40 agonistantibody. In some embodiments of any of the methods, the anti-human OX40agonist antibody increases effector T cell proliferation, relative toeffector T cell proliferation prior to administration of the OX40agonist antibody. In some embodiments of any of the methods, theanti-human OX40 agonist antibody increases memory T cell proliferation,relative to memory T cell proliferation prior to administration of theOX40 agonist antibody. In some embodiments of any of the methods, theanti-human OX40 agonist antibody increases effector T cell cytokineproduction (e.g., gamma interferon production), relative to effector Tcell cytokine production prior to administration of the OX40 agonistantibody. In some embodiments of any of the methods, the anti-human OX40agonist antibody increases memory T cell cytokine production (e.g.,gamma interferon production), relative to memory T cell cytokineproduction prior to administration of the OX40 agonist antibody. In someembodiments of any of the methods, the anti-human OX40 agonist antibodyincreases CD4+ effector T cell proliferation and/or CD8+ effector T cellproliferation relative to CD4+ effector T cell proliferation and/or CD8+effector T cell proliferation prior to administration of the OX40agonist antibody. In some embodiments of any of the methods, theanti-human OX40 agonist antibody increases memory T cell proliferation(e.g., CD4+ memory T cell proliferation), relative to memory T cellproliferation prior to administration of the OX40 agonist antibody. Insome embodiments, the CD4+ effector T cells in the individual haveenhanced proliferation, cytokine secretion and/or cytolytic activityrelative to proliferation, cytokine secretion and/or cytolytic activityprior to the administration of the anti-human OX40 agonist antibody.

In some embodiments of any of the methods of the invention, the numberof CD4+ effector T cells is elevated relative to prior to administrationof the anti-human OX40 agonist antibody. In some embodiments, CD4+effector T cell cytokine secretion is elevated relative to prior toadministration of the anti-human OX40 agonist antibody. In someembodiments of any of the methods, the CD8+ effector T cells in theindividual have enhanced proliferation, cytokine secretion and/orcytolytic activity relative to prior to the administration of theanti-human OX40 agonist antibody. In some embodiments, the number ofCD8+ effector T cells is elevated relative to prior to administration ofthe anti-human OX40 agonist antibody. In some embodiments, CD8+ effectorT cell cytokine secretion is elevated relative to prior toadministration of the anti-human OX40 agonist antibody.

In some embodiments of any of the methods of the invention, theanti-human OX40 agonist antibody binds human effector cells, e.g., bindsFcγR expressed by human effector cells. In some embodiments, the humaneffector cell performs ADCC effector function. In some embodiments, thehuman effector cell performs phagocytosis effector function.

In some embodiments of any of the methods of the invention, theanti-human OX40 agonist antibody comprising a variant IgG1 Fcpolypeptide comprising a mutation that eliminates binding to humaneffector cells (e.g., a DANA or N297G mutation) has diminished activity(e.g., CD4+ effector T cell function, e.g., proliferation), relative toanti-human OX40 agonist antibody comprising native sequence IgG1 Fcportion. In some embodiment, the anti-human OX40 agonist antibodycomprising a variant IgG1 Fc polypeptide comprising a mutation thateliminates binding to human effector cells (e.g., a DANA or N297Gmutation) does not possess substantial activity (e.g., CD4+ effector Tcell function, e.g., proliferation).

In some embodiments of any of the methods of the invention, antibodycross-linking is required for anti-human OX40 agonist antibody function.In some embodiments, function is stimulation of CD4+ effector T cellproliferation. In some embodiments, antibody cross-linking is determinedby providing anti-human OX40 agonist antibody adhered on a solid surface(e.g., a cell culture plate). In some embodiments, antibodycross-linking is determined by introducing a mutation in the antibody'sIgG1 Fc portion (e.g., a DANA or N297S mutation) and testing function ofthe mutant antibody.

In some embodiments of any of the methods, the memory T cells in theindividual have enhanced proliferation and/or cytokine secretionrelative to prior to the administration of the anti-human OX40 agonistantibody. In some embodiments, the number of memory T cells is elevatedrelative to prior to administration of the anti-human OX40 agonistantibody. In some embodiments, memory T cell cytokine secretion (level)is elevated relative to prior to administration of the anti-human OX40agonist antibody. In some embodiments of any of the methods, the Treg inthe individual have decreased inhibition of effector T cell function(e.g., proliferation and/or cytokine secretion) relative to prior to theadministration of the anti-human OX40 agonist antibody. In someembodiments, the number of effector T cells is elevated relative toprior to administration of the anti-human OX40 agonist antibody. In someembodiments, effector T cell cytokine secretion (level) is elevatedrelative to prior to administration of the anti-human OX40 agonistantibody.

In some embodiments of any of the methods of the invention, the numberof intratumoral (infiltrating) CD4+ effector T cells (e.g., total numberof CD4+ effector T cells, or e.g., percentage of CD4+ cells in CD45+cells) is elevated relative to prior to administration of the anti-humanOX40 agonist antibody. In some embodiments of any of the methods of theinvention, number of intratumoral (infiltrating) CD4+ effector T cellsthat express gamma interferon (e.g., total gamma interferon expressingCD4+ cells, or e.g., percentage of gamma interferon expressing CD4+cells in total CD4+ cells) is elevated relative to prior toadministration anti-human OX40 agonist antibody.

In some embodiments of any of the methods of the invention, the numberof intratumoral (infiltrating) CD8+ effector T cells (e.g., total numberof CD8+ effector T cells, or e.g., percentage of CD8+ in CD45+ cells) iselevated relative to prior to administration of anti-human OX40 agonistantibody. In some embodiments of any of the methods of the invention,number of intratumoral (infiltrating) CD8+ effector T cells that expressgamma interferon (e.g., percentage of CD8+ cells that express gammainterferon in total CD8+ cells) is increased relative to prior toadministration of anti-human OX40 agonist antibody.

In some embodiments of any of the methods of the invention, the numberof intratumoral (infiltrating) Treg (e.g., total number of Treg or e.g.,percentage of Fox3p+ cells in CD4+ cells) is reduced relative to priorto administration of anti-human OX40 agonist antibody.

In some embodiments of any of the methods of the invention,administration of anti-human OX40 agonist antibody is in combinationwith administration of a tumor antigen. In some embodiments, the tumorantigen comprises protein. In some embodiments, the tumor antigencomprises nucleic acid. In some embodiments, the tumor antigen is atumor cell.

In some embodiments of any of the methods of the invention, the cancerdisplays human effector cells (e.g., is infiltrated by human effectorcells). Methods for detecting human effector cells are well known in theart, including, e.g., by IHC. In some embodiments, the cancer displayhigh levels of human effector cells. In some embodiments, human effectorcells are one or more of NK cells, macrophages, monocytes. In someembodiments, the cancer is any cancer described herein.

In some embodiments of any of the methods of the invention, the cancerdisplays cells expressing FcR (e.g., is infiltrated by cells expressingFcR). Methods for detecting FcR are well known in the art, including,e.g., by IHC. In some embodiments, the cancer display high levels ofcells expressing FcR. In some embodiments, FcR is FcγR. In someembodiments, FcR is activating FcγR.

An “individual” according to any of the above embodiments is preferablya human.

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, a combination therapy ofthe invention (e.g., including an OX40 binding agonist and ananti-angiogenesis agent) may be co-administered with at least oneadditional therapeutic agent.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent or agents. In one embodiment, administration of theanti-OX40 antibody and anti-angiogenesis agent (e.g., anti-VEGFantibodies) and administration of an additional therapeutic agent occurwithin about one month, or within about one, two or three weeks, orwithin about one, two, three, four, five, or six days, of each other.Antibodies of the invention can also be used in combination withradiation therapy.

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with a chemotherapy or chemotherapeutic agent. In someembodiments, an anti-human OX40 agonist antibody may be administered inconjunction with a radiation therapy or radiotherapeutic agent. In someembodiments, an anti-human OX40 agonist antibody may be administered inconjunction with a targeted therapy or targeted therapeutic agent. Insome embodiments, an anti-human OX40 agonist antibody may beadministered in conjunction with an immunotherapy or immunotherapeuticagent, for example a monoclonal antibody.

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with a PARP inhibitor (e.g., Olaparanib, Rucaparib,Niraparib, Cediranib, BMN673, Veliparib), Trabectedin, nab-paclitaxel(albumen-bound paclitaxel, ABRAXANE), Trebananib, Pazopanib, Cediranib,Palbociclib, everolimus, fluoropyrimidine (e.g., FOLFOX, FOLFIRI), IFL,regorafenib, Reolysin, Alimta, Zykadia, Sutent, Torisel (temsirolimus),Inlyta (axitinib, Pfizer), Afinitor (everolimus, Novartis), Nexavar(sorafenib, Onyx/Bayer), Votrient, Pazopanib, axitinib, IMA-901,AGS-003, cabozantinib, Vinflunine, Hsp90 inhibitor (e.g., apatorsin),Ad-GM-CSF (CT-0070), Temazolomide, IL-2, IFNa, vinblastine, Thalomid,dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide,bortezomid (VELCADE), amrubicine, carfilzomib, pralatrexate, and/orenzastaurin.

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with a PD-1 axis binding antagonist. A PD-1 axis bindingantagonist includes but is not limited to a PD-1 binding antagonist, aPD-L1 binding antagonist and a PD-L2 binding antagonist. Alternativenames for “PD-1” include CD279 and SLEB2. Alternative names for “PD-L1”include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PD-L2”include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PD-L1, andPD-L2 are human PD-1, PD-L1 and PD-L2. In some embodiments, the PD-1binding antagonist is a molecule that inhibits the binding of PD-1 toits ligand binding partners. In a specific aspect the PD-1 ligandbinding partners are PD-L1 and/or PD-L2. In another embodiment, a PD-L1binding antagonist is a molecule that inhibits the binding of PD-L1 toits binding partners. In a specific aspect, PD-L1 binding partners arePD-1 and/or B7-1. In another embodiment, the PD-L2 binding antagonist isa molecule that inhibits the binding of PD-L2 to its binding partners.In a specific aspect, a PD-L2 binding partner is PD-1. The antagonistmay be an antibody, an antigen binding fragment thereof, animmunoadhesin, a fusion protein, or oligopeptide. In some embodiments,the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a humanantibody, a humanized antibody, or a chimeric antibody). In someembodiments, the anti-PD-1 antibody is selected from the groupconsisting of MDX-1106 (nivolumab, OPDIVO), Merck 3475 (MK-3475,pembrolizumab, KEYTRUDA) and CT-011 (Pidilizumab). In some embodiments,the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesincomprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2fused to a constant region (e.g., an Fc region of an immunoglobulinsequence). In some embodiments, the PD-1 binding antagonist is AMP-224.In some embodiments, the PD-L1 binding antagonist is anti-PD-L1antibody. In some embodiments, the anti-PD-L1 binding antagonist isselected from the group consisting of YW243.55.S70, MPDL3280A, MEDI4736and MDX-1105. MDX-1105, also known as BMS-936559, is an anti-PD-L1antibody described in WO2007/005874. Antibody YW243.55.S70 (heavy andlight chain variable region sequences shown in SEQ ID Nos. 20 and 21,respectively) is an anti-PD-L1 described in WO 2010/077634 A1. MDX-1106,also known as MDX-1106-04, ONO-4538, BMS-936558 or nivolumab, is ananti-PD-1 antibody described in WO2006/121168. Merck 3475, also known asMK-3475, SCH-900475 or pembrolizumab, is an anti-PD-1 antibody describedin WO2009/114335. CT-011, also known as hBAT, hBAT-1 or pidilizumab, isan anti-PD-1 antibody described in WO2009/101611. AMP-224, also known asB7-DCIg, is a PD-L2-Fc fusion soluble receptor described inWO2010/027827 and WO201 1/066342. In some embodiments, the anti-PD-1antibody is MDX-1106. Alternative names for “MDX-1106” include MDX-1106-04, ONO-4538, BMS-936558 or nivolumab. In some embodiments, theanti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4).

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an agonist directed against an activatingco-stimulatory molecule. In some embodiments, an activatingco-stimulatory molecule may include CD40, CD226, CD28, GITR, CD137,CD27, HVEM, or CD127. In some embodiments, the agonist directed againstan activating co-stimulatory molecule is an agonist antibody that bindsto CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an antagonist directed against an inhibitoryco-stimulatory molecule. In some embodiments, an inhibitoryco-stimulatory molecule may include CTLA-4 (also known as CD152), PD-1,TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, orarginase. In some embodiments, the antagonist directed against aninhibitory co-stimulatory molecule is an antagonist antibody that bindsto CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3 (e.g., LAG-3-IgG fusionprotein (IMP321)), B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase.

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an antagonist directed against CTLA-4 (also known asCD152), e.g., a blocking antibody. In some embodiments, an anti-humanOX40 agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with ipilimumab (also knownas MDX-010, MDX-101, or Yervoy®). In some embodiments, an anti-humanOX40 agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with tremelimumab (alsoknown as ticilimumab or CP-675,206). In some embodiments, an anti-humanOX40 agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with an antagonist directedagainst B7-H3 (also known as CD276), e.g., a blocking antibody. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with MGA271. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with an antagonist directedagainst a TGF beta, e.g., metelimumab (also known as CAT-192),fresolimumab (also known as GC1008), or LY2157299.

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with a treatment comprising adoptive transfer of a T cell(e.g., a cytotoxic T cell or CTL) expressing a chimeric antigen receptor(CAR). In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with UCART19. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with WT128z. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with KTE-C19 (Kite). In some embodiments, an anti-humanOX40 agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with CTL019 (Novartis). Insome embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with a treatment comprising adoptive transfer of a T cellcomprising a dominant-negative TGF beta receptor, e.g, adominant-negative TGF beta type II receptor. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with a treatmentcomprising a HERCREEM protocol (see, e.g., ClinicalTrials.gov IdentifierNCT00889954).

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an antagonist directed against CD19. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with MOR00208. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with an antagonist directedagainst CD38. In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with daratumumab.

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an agonist directed against CD137 (also known asTNFRSF9, 4-1BB, or ILA), e.g., an activating antibody. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with urelumab (also known as BMS-663513). In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an agonist directed against CD40, e.g., anactivating antibody. In some embodiments, an anti-human OX40 agonistantibody and/or anti-angiogenesis agent (e.g., anti-VEGF antibody) maybe administered in conjunction with CP-870893. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with an agonistdirected against OX40 (also known as CD134), e.g., an activatingantibody. In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with a different anti-OX40 antibody (e.g.,AgonOX). In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an agonist directed against CD27, e.g., anactivating antibody. In some embodiments, an anti-human OX40 agonistantibody and/or anti-angiogenesis agent (e.g., anti-VEGF antibody) maybe administered in conjunction with CDX-1127. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with anantagonist directed against indoleamine-2,3-dioxygenase (IDO). In someembodiments, with the IDO antagonist is 1-methyl-D-tryptophan (alsoknown as 1-D-MT).

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an agonist directed against CD137 (also known asTNFRSF9, 4-1BB, or ILA), e.g., an activating antibody. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with urelumab (also known as BMS-663513). In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an agonist directed against CD40, e.g., anactivating antibody. In some embodiments, an anti-human OX40 agonistantibody and/or anti-angiogenesis agent (e.g., anti-VEGF antibody) maybe administered in conjunction with CP-870893 or R07009789. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an agonist directed against OX40 (also known asCD134), e.g., an activating antibody.). In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with an agonistdirected against CD27, e.g., an activating antibody. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with CDX-1127 (also known as varlilumab). In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an antagonist directed againstindoleamine-2,3-dioxygenase (IDO). In some embodiments, with the IDOantagonist is 1-methyl-D-tryptophan (also known as 1-D-MT). In someembodiments, the IDO antagonist is an IDO antagonist shown inWO2010/005958 (the contents of which are expressly incorporated byrecord herein). In some embodiments the IDO antagonist is4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide(e.g., as described in Example 23 of WO2010/005958). In some embodimentsthe IDO antagonist is

In some embodiments, the IDO antagonist is 1NCB24360. In someembodiments, the IDO antagonist is Indoximod (the D isomer of1-methyl-tryptophan). In some embodiments, an anti-human OX40 agonistantibody and/or anti-angiogenesis agent (e.g., anti-VEGF antibody) maybe administered in conjunction with an antibody-drug conjugate. In someembodiments, the antibody-drug conjugate comprises mertansine ormonomethyl auristatin E (MMAE). In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with an anti-NaPi2bantibody-MMAE conjugate (also known as DNIB0600A, RG7599 or lifastuzumabvedotin). In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with trastuzumab emtansine (also known asT-DM 1, ado-trastuzumab emtansine, or KADCYLA®, Genentech). In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an anti-MUC16 antibody-MMAE conjugate, DMUC5754A. Insome embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an anti-MUC16 antibody-MMAE conjugate, DMUC4064A. Insome embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an antibody-drug conjugate targeting the endothelinB receptor (EDNBR), e.g., an antibody directed against EDNBR conjugatedwith MMAE. In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with an antibody-drug conjugate targetingthe lymphocyte antigen 6 complex, locus E (Ly6E), e.g., an antibodydirected against Ly6E conjugated with MMAE, (also known as DLYE5953A).In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with polatuzumab vedotin. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with anantibody-drug conjugate targeting CD30. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with ADCETRIS(also known as brentuximab vedotin). In some embodiments, an anti-humanOX40 agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with polatuzumab vedotin.

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an angiogenesis inhibitor. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with an antibodydirected against a VEGF, e.g., VEGF-A. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with bevacizumab(also known as AVASTIN®, Genentech). In some embodiments, an anti-humanOX40 agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with an antibody directedagainst angiopoietin 2 (also known as Ang2). In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with MEDI3617. Insome embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an antibody directed against VEGFR2. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with ramucirumab. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with a VEGF Receptor fusionprotein. In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with aflibercept. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with ziv-aflibercept (alsoknown as VEGF Trap or Zaltrap®). In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with a bispecific antibodydirected against VEGF and Ang2. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with RG7221 (also known asvanucizumab). In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with an angiogenesis inhibitor and inconjunction with a PD-1 axis binding antagonist (e.g., a PD-1 bindingantagonist such as an anti-PD-1 antibody, a PD-L1 binding antagonistsuch as an anti-PD-L1 antibody, and a PD-L2 binding antagonist such asan anti-PD-L2 antibody). In some embodiments, an anti-human OX40 agonistantibody and/or anti-angiogenesis agent (e.g., anti-VEGF antibody) maybe administered in conjunction with bevacizumab and a PD-1 axis bindingantagonist (e.g., a PD-1 binding antagonist such as an anti-PD-1antibody, a PD-L1 binding antagonist such as an anti-PD-L1 antibody, anda PD-L2 binding antagonist such as an anti-PD-L2 antibody). In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with bevacizumab and MDX-1106 (nivolumab, OPDIVO). Insome embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with bevacizumab and Merck 3475 (MK-3475, pembrolizumab,KEYTRUDA). In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with bevacizumab and CT-011 (Pidilizumab).In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with bevacizumab and YW243.55.S70. In some embodiments,an anti-human OX40 agonist antibody and/or anti-angiogenesis agent(e.g., anti-VEGF antibody) may be administered in conjunction withbevacizumab and MPDL3280A. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with bevacizumab andMEDI4736. In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with bevacizumab and MDX-1105.

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an antineoplastic agent. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with an agenttargeting CSF-1R (also known as M-CSFR or CD115). In some embodiments,an anti-human OX40 agonist antibody and/or anti-angiogenesis agent(e.g., anti-VEGF antibody) may be administered in conjunction withanti-CSF-1R antibody (also known as IMC-CS4 or LY3022855) In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with anti-CSF-1R antibody, RG7155 (also known asR05509554 or emactuzumab). In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with an interferon, forexample interferon alpha or interferon gamma. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with Roferon-A(also known as recombinant Interferon alpha-2a). In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with GM-CSF (alsoknown as recombinant human granulocyte macrophage colony stimulatingfactor, rhu GM-CSF, sargramostim, or Leukine®). In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with IL-2 (alsoknown as aldesleukin or Proleukin®). In some embodiments, an anti-humanOX40 agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with IL-12. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with IL27. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with IL-15. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with ALT-803. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with an antibody targetingCD20. In some embodiments, the antibody targeting CD20 is obinutuzumab(also known as GA101 or Gazyva®) or rituximab. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with an antibodytargeting GITR. In some embodiments, the antibody targeting GITR isTRX518. In some embodiments, the antibody targeting GITR is MK04166(Merck).

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an inhibitor of Bruton's tyrosine kinase (BTK). Insome embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with ibrutinib. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with an inhibitor ofIsocitrate dehydrogenase 1 (IDH1) and/or Isocitrate dehydrogenase 2(IDH2). In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with AG-120 (Agios).

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with obinutuzumab and a PD-1 axis binding antagonist(e.g., a PD-1 binding antagonist such as an anti-PD-1 antibody, a PD-L1binding antagonist such as an anti-PD-L1 antibody, and a PD-L2 bindingantagonist such as an anti-PD-L2 antibody).

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with a cancer vaccine. In some embodiments, the cancervaccine is a peptide cancer vaccine, which in some embodiments is apersonalized peptide vaccine. In some embodiments the peptide cancervaccine is a multivalent long peptide, a multi-peptide, a peptidecocktail, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine(see, e.g., Yamada et al., Cancer Sci, 104:14-21, 2013). In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an adjuvant. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with a treatment comprisinga TLR agonist, e.g., Poly-ICLC (also known as Hiltonol®), LPS, MPL, orCpG ODN. In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with tumor necrosis factor (TNF) alpha. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with IL-1. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with HMGB1. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an IL-10 antagonist. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with an IL-4antagonist. In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with an IL-13 antagonist. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an IL-17 antagonist. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with an HVEMantagonist. In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with an ICOS agonist, e.g., byadministration of ICOS-L, or an agonistic antibody directed againstICOS. In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with a treatment targeting CX3CL1. In some embodiments,an anti-human OX40 agonist antibody and/or anti-angiogenesis agent(e.g., anti-VEGF antibody) may be administered in conjunction with atreatment targeting CXCL9. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with a treatment targetingCXCL10. In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with a treatment targeting CCL5. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with an LFA-1 orICAM1 agonist. In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with a Selectin agonist.

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an inhibitor of B-Raf. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with vemurafenib(also known as Zelboraf®). In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with dabrafenib (also knownas Tafinlar®). In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with encorafenib (LGX818).

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an EGFR inhibitor. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with erlotinib(also known as Tarceva®). In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with an inhibitor ofEGFR-T790M. In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with gefitinib. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with afatinib. Insome embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with cetuximab (also known as Erbitux®). In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with panitumumab (also known as Vectibix®). In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with rociletinib. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with AZD9291. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an inhibitor of a MEK, such as MEK1 (also known asMAP2K1) and/or MEK2 (also known as MAP2K2). In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with cobimetinib(also known as GDC-0973 or XL-518). In some embodiments, an anti-humanOX40 agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with trametinib (also knownas Mekinist®). In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with binimetinib.

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction an inhibitor of B-Raf (e.g., vemurafenib or dabrafenib)and an inhibitor of MEK (e.g., MEK1 and/or MEK2 (e.g., cobimetinib ortrametinib). In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with an inhibitor of ERK (e.g., ERK1/2). Insome embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with GDC-0994). In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with an inhibitor of B-Raf,an inhibitor of MEK, and an inhibitor of ERK1/2. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with an inhibitorof EGFR, an inhibitor of MEK, and an inhibitor of ERK1/2. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with one or more MAP kinase pathway inhibitor. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with CK127. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with an inhibitor of K-Ras.

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an inhibitor of c-Met. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with onartuzumab(also known as MetMAb). In some embodiments, an anti-human OX40 agonistantibody and/or anti-angiogenesis agent (e.g., anti-VEGF antibody) maybe administered in conjunction with an inhibitor of anaplatic lymphomakinase (ALK). In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with AF802 (also known as CH5424802 oralectinib). In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with crizotinib. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with ceritinib.In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an inhibitor of a phosphatidylinositol 3-kinase(PI3K). In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjuction with buparlisib (BKM-120). In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with pictilisib(also known as GDC-0941). In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with buparlisib (also knownas BKM-120). In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with perifosine (also known as KRX-0401). Insome embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with a delta-selective inhibitor of aphosphatidylinositol 3-kinase (PI3K). In some embodiments, an anti-humanOX40 agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with idelalisib (also knownas GS-1101 or CAL-101). In some embodiments, an anti-human OX40 agonistantibody and/or anti-angiogenesis agent (e.g., anti-VEGF antibody) maybe administered in conjunction with taselisib (also known as GDC-0032).In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with BYL-719. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with an inhibitor of anAkt. In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with MK2206. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with GSK690693. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with ipatasertib (also known as GDC-0068). In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an inhibitor of mTOR. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with sirolimus(also known as rapamycin). In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with temsirolimus (alsoknown as CCI-779 or Torisel®). In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with everolimus (also knownas RAD001). In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with ridaforolimus (also known as AP-23573,MK-8669, or deforolimus). In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with OSI-027. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with AZD8055. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with INK128. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with a dual PI3K/mTOR inhibitor. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with XL765. Insome embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with GDC-0980. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with BEZ235 (also known asNVP-BEZ235). In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with BGT226. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with GSK2126458.In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with PF-04691502. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with PF-05212384 (alsoknown as PKI-587).

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an agent that selectively degrades the estrogenreceptor. In some embodiments, an anti-human OX40 agonist antibodyand/or anti-angiogenesis agent (e.g., anti-VEGF antibody) may beadministered in conjunction with GDC-0927. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with an inhibitorof HER3. In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with duligotuzumab. In some embodiments, an anti-humanOX40 agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with an inhibitor of LSD 1.In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an inhibitor of MDM2. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with an inhibitorof BCL2. In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with venetoclax. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with an inhibitor of CHK1.In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with GDC-0575. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with an inhibitor ofactivated hedgehog signaling pathway. In some embodiments, an anti-humanOX40 agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with ERIVEDGE.

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with radiation therapy. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with gemcitabine.In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with nab-paclitaxel (ABRAXANE). In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with trastuzumab.In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with TVEC. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with IL27. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with cyclophosphamide. In some embodiments, an anti-humanOX40 agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with an agent that recruitsT cells to the tumor. In some embodiments, an anti-human OX40 agonistantibody and/or anti-angiogenesis agent (e.g., anti-VEGF antibody) maybe administered in conjunction with lirilumab (IPH2102/BMS-986015). Insome embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with Idelalisib. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with an antibody thattargets CD3 and CD20. In some embodiments, an anti-human OX40 agonistantibody and/or anti-angiogenesis agent (e.g., anti-VEGF antibody) maybe administered in conjunction with REGN1979. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with an antibodythat targets CD3 and CD19. In some embodiments, an anti-human OX40agonist antibody and/or anti-angiogenesis agent (e.g., anti-VEGFantibody) may be administered in conjunction with blinatumomab.

In some embodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with an oncolytic virus. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with carboplatinand nab-paclitaxel. In some embodiments, an anti-human OX40 agonistantibody and/or anti-angiogenesis agent (e.g., anti-VEGF antibody) maybe administered in conjunction with carboplatin and paclitaxel. In someembodiments, an anti-human OX40 agonist antibody and/oranti-angiogenesis agent (e.g., anti-VEGF antibody) may be administeredin conjunction with cisplatin and pemetrexed. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with cisplatinand gemcitabine. In some embodiments, an anti-human OX40 agonistantibody and/or anti-angiogenesis agent (e.g., anti-VEGF antibody) maybe administered in conjunction with FOLFOX. In some embodiments, ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (e.g.,anti-VEGF antibody) may be administered in conjunction with FOLFIRI.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the anti-human OX40 agonist antibody and/oranti-angiogenesis agent can occur prior to, simultaneously, and/orfollowing, administration of the additional therapeutic agent and/oradjuvant. Anti-human OX40 agonist antibodies and/or anti-angiogenesisagents can also be used in combination with radiation therapy.

An anti-human OX40 agonist antibody and/or anti-angiogenesis agent (andany additional therapeutic agent) can be administered by any suitablemeans, including parenteral, intrapulmonary, and intranasal, and, ifdesired for local treatment, intralesional administration. Parenteralinfusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. Dosing can be by anysuitable route, e.g. by injections, such as intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic. Various dosing schedules including but not limited to single ormultiple administrations over various time-points, bolus administration,and pulse infusion are contemplated herein.

Anti-human OX40 agonist antibodies and anti-angiogenesis agents would beformulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The antibody need not be, but isoptionally formulated with one or more agents currently used to preventor treat the disorder in question. The effective amount of such otheragents depends on the amount of antibody present in the formulation, thetype of disorder or treatment, and other factors discussed above. Theseare generally used in the same dosages and with administration routes asdescribed herein, or about from 1 to 99% of the dosages describedherein, or in any dosage and by any route that is empirically/clinicallydetermined to be appropriate. Further, exemplary dosages for ananti-angiogenesis agent (e.g., an anti-VEGF antibody) are providedbelow.

For the prevention or treatment of disease, the appropriate dosage of ananti-human OX40 agonist antibody and/or anti-angiogenesis agent (whenused alone or in combination with one or more other additionaltherapeutic agents) will depend on the type of disease to be treated,the type of antibody, the severity and course of the disease, whetherthe antibody is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The antibody issuitably administered to the patient at one time or over a series oftreatments. Depending on the type and severity of the disease, about 1μg/kg to 40 mg/kg of antibody can be an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. Such doses may be administered intermittently, e.g. every weekor every three weeks (e.g. such that the patient receives from about twoto about twenty, or e.g. about six doses of the antibody). An initialhigher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-human OX40 agonist antibody and/oranti-angiogenesis agent.

Exemplary doses for anti-VEGF antibodies are provided below. It will beappreciated by one of skill in the art that these doses are merelyexemplary and are based on dosing of anti-VEGF antibody alone. Dosingand/or administration practices described herein for anti-VEGF antibodytreatment alone may of course be modified when combined with OX40binding agonist treatment. In some embodiments, the OX40 binding agonistis administered before the anti-angiogenesis agent (e.g., anti-VEGFantibody), simultaneous with the anti-angiogenesis agent, or after theanti-angiogenesis agent.

For the prevention or treatment of cancer, the dose of VEGF antagonist(e.g., an anti-VEGF antibody, such as bevacizumab), anti-human OX40agonist antibody, and/or chemotherapeutic agent will depend on the typeof cancer to be treated, as defined above, the severity and course ofthe cancer, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the drug, and the discretion of the attending physician.In one embodiment, VEGF antagonist (e.g., bevacizumab) is administeredat 5 mg/kg of body weight given once every 2 weeks, 10 mg/kg of bodyweight given once every 2 weeks, 7.5 mg/kg of body weight given onceevery 3 weeks, or 15 mg/kg of body weight given once every 3 weeks.

With respect to bevacizumab for the treatment of colorectal cancer, thepreferred dosages according to the EMEA are 5 mg/kg or 10 mg/kg of bodyweight given once every 2 weeks or 7.5 mg/kg or 15 mg/kg of body weightgiven once every 3 weeks. For the treatment of NSCLC, the preferreddosage is 15 mg/kg given once every 3 weeks by infusion in combinationwith carboplatin and paclitaxel. For the treatment of renal cellcarcinoma, the preferred dosage is 10 mg/kg given once every 2 weeks byinfusion with interferon a-2a or as a monotherapy. For the treatment ofcervical cancer, the preferred dosage is 15 mg/kg given once every threeweeks by infusion and administered in combination with one of thefollowing chemotherapy regimens: paclitaxel and cisplatin or paclitaxeland topotecan. For the treatment of glioblastoma, the preferred dosageis 10 mg/kg given once every two weeks by infusion.

In one embodiment, a fixed dose of the VEGF antagonist is administered.A “fixed” or “flat” dose of a therapeutic agent herein refers to a dosethat is administered to a human patient without regard for the weight(WT) or body surface area (BSA) of the patient. The fixed or flat doseis therefore not provided as a mg/kg dose or a mg/m² dose, but rather asan absolute amount of the therapeutic agent. The fixed dose may suitablybe administered to the patient at one time or over a series oftreatments. Where a fixed dose is administered, preferably it is in therange from about 20 mg to about 2000 mg of the inhibitor. For example,the fixed dose may be approximately 420 mg, approximately 525 mg,approximately 840 mg, or approximately 1050 mg of the inhibitor (e.g.,an anti-VEGF antibody, such as bevacizumab). Where a series of doses areadministered, these may, for example, be administered approximatelyevery week, approximately every 2 weeks, approximately every 3 weeks, orapproximately every 4 weeks, but preferably approximately every 3 weeks.The fixed doses may, for example, continue to be administered untildisease progression, adverse event, or other time as determined by thephysician. For example, from about two, three, or four, up to about 17or more fixed doses may be administered.

Administration of an angiogenesis inhibitor, e.g., an anti-VEGFantibody, such as bevacizumab, and/or a phannaceuticalcomposition/treatment regimen comprising an angiogenesis inhibitor,e.g., an anti-VEGF antibody, such as bevacizumab, to a patient in needof such treatment or medical intervention may be by any suitable meansknown in the art for administration of a therapeutic antibody.Nonlimiting routes of administration include by oral, intravenous,intraperitoneal, subcutaneous, intramuscular, topical, intradermal,intranasal or intrabronchial administration (for example as effected byinhalation). Particularly preferred in context of this invention isparenteral administration, e.g., intravenous administration Where a VEGFantagonist is administered as a “single anti-tumor agent” it is the onlyanti-tumor agent administered to treat the cancer. i.e., it is notadministered in combination with another anti-tumor agent, such aschemotherapy or an OX40 binding agonist.

In one embodiment, one or more loading dose(s) of the VEGF antagonist(e.g., an anti-VEGF antibody, such as bevacizumab) are administered,followed by one or more maintenance dose(s). A “loading” dose hereingenerally comprises an initial dose of a therapeutic agent administeredto a patient, and is followed by one or more maintenance dose(s)thereof. Generally, a single loading dose is administered, but multipleloading doses are contemplated herein. Usually, the amount of loadingdose(s) administered exceeds the amount of the maintenance dose(s)administered and/or the loading dose(s) are administered more fiequentlythan the maintenance dose(s), so as to achieve the desired steady-stateconcentration of the therapeutic agent earlier than can be achieved withthe maintenance dose(s). A “maintenance” dose or “extended” dose hereinrefers to one or more doses of a therapeutic agent administered to thepatient over a treatment period. Usually, the maintenance doses areadministered at spaced treatment intervals, such as approximately everyweek, approximately every 2 weeks, approximately every 3 weeks, orapproximately every 4 weeks. In another embodiment, a plurality of thesame dose is administered to the patient. According to one preferredembodiment of the invention, a fixed dose of a VEGF antagonist (e.g., ananti-VEGF antibody, such as bevacizumab) of approximately 840 mg(loading dose) is administered, followed by one or more doses ofapproximately 420 mg (maintenance dose(s)) of the antagonist. Themaintenance doses are preferably administered about every 3 weeks, for atotal of at least two doses, up to 17 or more doses.

According to another preferred embodiment of the invention, one or morefixed dose(s) of approximately 1050 mg of the VEGF antagonist (e.g., ananti-VEGF antibody, such as bevacizumab) are administered, for exampleevery 3 weeks. According to this embodiment, one, two or more of thefixed doses are administered, e.g., for up to one year (17 cycles), andlonger as desired.

In another embodiment, a fixed dose of approximately 1050 mg of the VEGFantagonist (e.g., an anti-VEGF antibody, such as bevacizumab) isadministered as a loading dose, followed by one or more maintenancedose(s) of approximately 525 mg. About one, two, or more maintenancedoses may be administered to the patient every 3 weeks according to thisembodiment.

While the VEGF antagonist (e.g., an anti-VEGF antibody, such asbevacizumab) and/or anti-human OX40 agonist antibody or chemotherapeuticagent may be administered in conjunction, the patient is optionallytreated with a combination of the inhibitor (or chemotherapeutic agent),and one or more (additional) chemotherapeutic agent(s). Exemplarychemotherapeutic agents herein include: geincitabine, carboplatin,oxaliplatin, irinotecan, fluoropyrimidine (e.g., 5-FU), paclitaxel(e.g., nab-paclitaxel), docetaxel, topotecan, capecitabine,temozolomide, interferon-alpha, and/or liposomal doxorubicin (e.g.,pegylated liposomal doxorubicin). In some embodiments, at least one ofthe chemotherapeutic agents is carboplatin or paclitaxel. In someembodiments, at least one of the chemotherapeutic agents is carboplatinor gemcitabine. The combined administration includes co-administrationor concurrent administration, using separate formulations or a singlepharmaceutical formulation, and consecutive administration in eitherorder, wherein preferably there is a time period while both (or all)active agents simultaneously exert their biological activities. Thus,the chemotherapeutic agent may be adm inistered prior to, or following,administration of the VEGF antagonist (e.g., an anti-VEGF antibody, suchas bevacizumab). In this embodiment, the timing betw een at least oneadministration of the chemotherapeutic agent and at least oneadministration of the VEGF antagonist (e.g., an anti-VEGF antibody, suchas bevacizumab) is preferably approximately 1 month or less, and mostpreferably approximately 2 weeks or less. Alternatively, thechemotherapeutic agent and the inhibitor are administered concurrentlyto the patient, in a single formulation or separate formulations.Treatment with the combination of the chemotherapeutic agent (e.g.,carboplatin and/or paclitaxel) and the VEGF antagonist (e.g., ananti-VEGF antibody, such as bevacizumab) may result in a synergistic, orgreater than additive, therapeutic benefit to the patient.

Particularly desired chemotherapeutic agents for combining with the VEGFantagonist (e.g., an anti-VEGF antibody, such as bevacizumab) and/oranti-human OX40 agonist antibody, e.g. for therapy of ovarian cancer,include: a chemotherapeutic agent such as a platinum compound (e.g.,carboplatin), a taxol such as paclitaxel or docetaxel, topotecari, orliposomal doxorubicin.

Particularly desired chemotherapeutic agents for combining with the VEGFantagonist (e.g., an anti-VEGF antibody, such as bevacizumab) and/oranti-human OX40 agonist antibody, e.g., for therapy of advanced stageepithelial ovarian cancer, fallopian tube cancer, or primary peritonealcancer include: chemotherapeutic agents such as carboplatin, paclitaxel,and/or gemcitabine.

Particularly desired chemotherapeutic agents for combining with the VEGFantagonist (e.g., an anti-VEGF antibody such as bevacizumab) and/oranti-human OX40 agonist antibody, e.g., for therapy ofplatinum-sensitive epithelial ovarian cancer, fallopian tube cancer, orprimary peritoneal cancer include: chemotherapeutic agents such ascarboplatin and gemcitabine.

Particularly desired chemotherapeutic agents for combining with the VEGFantagonist (e.g., an anti-VEGF antibody such as bevacizumab) and/oranti-human OX40 agonist antibody, e.g., for therapy ofplatinum-resistant recurrent epithelial ovarian cancer, fallopian tubecancer, or primary peritoneal cancer include: a chemotherapeutic agentsuch as paclitaxel, topotecan, or pegylated liposomal doxorubicin.

Particularly desired chemotherapeutic agents for combining with the VEGFantagonist (e.g., an anti-VEGF antibody, such as bevacizumab) and/oranti-human OX40 agonist antibody, e.g., for therapy of breast cancer,include: chemotherapeutic agents such as capecitabine, and a taxol suchas paclitaxel (e.g., nab-paclitaxel) or docetaxel.

Particularly desired chemotherapeutic agents for combining with the VEGFantagonist (e.g., an anti-VEGF antibody, such as bevacizumab) and/oranti-human OX40 agonist antibody, e.g., for therapy of glioblastoma,include: chemotherapeutic agents such as temozolomide, optionally incombination with radiotherapy.

Particularly desired chemotherapeutic agents for combining with the VEGFantagonist (e.g., an anti-VEGF antibody, such as bevacizumab) and/oranti-human OX40 agonist antibody, e.g., for therapy of colorectalcancer, include: chemotherapeutic agents such as a fluoropyrimidine(e.g., 5-FU), paclitaxel, cisplatin, topotecan, irinotecan,fluoropyrimidine-oxaliplatin, fluoropyrimidine-irinotecan. FOLFOX4 (5-FUlecovorin, oxaliplatin), and IFL (ironotecan, 5-FU, leucovorin).

Particularly desired chemotherapeutic agents for combining with the VEGFantagonist (e.g., an anti-VEGF antibody, such as bevacizumab) and/oranti-human OX40 agonist antibody, e.g., for therapy of renal cellcarcinoma, include: chemotherapeutic agents such as interferon-alpha2a.

Particularly desired chemotherapeutic agents for combining with the VEGFantagonist (e.g., an anti-VEGF antibody, such as bevacizumab) and/oranti-human OX40 agonist antibody, e.g., for therapy of cervical cancer,include: chemotherapeutic agents such as paclitaxel, cisplatin,topotecan, paclitaxel in combination with cispiatin, and paclitaxel incombination with topotecan.

A chernotherapeutic agent, if administered, is usually administered atdosages known therefore, or optionally lowered due to combined action ofthe drugs or negative side effects attributable to administration of thechemotherapeutic agent. Preparation and dosing schedules for suchchemotherapeutic agents may be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Where the chemotherapeutic agent is paclitaxel, preferably, it isadministered at a dose between about 130 mg/m² to 200 mg/m² (for exampleapproximately 175 mg/m²), for instance, over 3 hours, once every 3weeks. Where the chemotherapeutic agent is carboplatin, preferably it isadministered by calculating the dose of carboplatin using the Calvertformula which is based on a patient's preexisting renal function orrenal function and desired platelet nadir. Renal excretion is the majorroute of elimination for carboplatin. The use of this dosing formula, ascompared to empirical dose calculation based on body surface area,allows compensation for patient variations in pretreatment renalfunction that might otherwise result in either underdosing (in patientswith above average renal function) or overdosing (in patients withimpaired renal function). The target AUC of 4-6 mg/mL/min using singleagent carboplatin appears to provide the most appropriate dose range inpreviously treated patients.

Aside from the VEGF antagonist (e.g., an anti-VEGF antibody, such asbevacizumab), anti-human OX40 agonist antibody, and chemotherapeuticagent, other therapeutic regimens may be combined therewith. Forexample, a second (third, fourth, etc.) chemotherapeutic agent(s) may beadministered, wherein the second chemotherapeutic agent is anantimetabolite chemotherapeutic agent, or a chemotherapeutic agent thatis not an antimetabolite. For example, the second chemotherapeutic agentmay be a taxane (such as paclitaxel or docetaxel), capecitabine, orplatinum-based chemotherapeutic agent (such as carboplatin, cisplatin,or oxaliplatin), anthracycline (such as doxorubicin, including,liposomal doxorubicin), topotecan, pemetrexed, vinca alkaloid (such asvinorelbine), and TLK 286. “Cocktails” of different chemotherapeuticagents may be administered.

Suitable dosages for any of the above-noted co-administered agents arethose presently used and may be lowered due to the combined action(synergy) of the agent and inhibitor. In addition to the abovetherapeutic regimes, the patient may be subjected to surgical removal oftumors and/or cancer cells, and/or radiation therapy.

Where the VEGF antagonist is an antibody (e.g., bevacizumab), preferablythe administered antibody is a naked antibody. The VEGF antagonist(e.g., an anti-VEGF antibody, such as bevacizumab) administered may beconjugated with a cytotoxic agent. Preferably, the conjugated and/orantigen to which it is bound is/are internalized by the cell, resultingin increased therapeutic efficacy of the conjugate in killing the cancercell to which it binds. In a preferred embodiment, the cytotoxic agenttargets or interferes with nucleic acid in the cancer cell. Examples ofsuch cytotoxic agents include maytansinoids, calicheaimicins,ribonucleases, and DNA endonucleases. VI. Articles of Manufacture orKits

In another aspect of the invention, an article of manufacture or kitcontaining materials useful for the treatment, prevention and/ordiagnosis of the disorders described above is provided. The article ofmanufacture or kit comprises a container and a label or package inserton or associated with the container. Suitable containers include, forexample, bottles, vials, syringes, IV solution bags, etc. The containersmay be formed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is by itself or combined withanother composition effective for treating, preventing and/or diagnosingthe condition and may have a sterile access port (for example thecontainer may be an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). At least one active agentin the composition is an antibody of the invention (e.g., an anti-humanOX40 agonist antibody of the present disclosure or an anti-angiogenicantibody of the present disclosure, such as an anti-VEGF antibody). Thelabel or package insert indicates that the composition is used fortreating the condition of choice. Moreover, the article of manufactureor kit may comprise (a) a first container with a composition containedtherein, wherein the composition comprises an antibody of the invention;and (b) a second container with a composition contained therein, whereinthe composition comprises a further cytotoxic or otherwise therapeuticagent. The article of manufacture in this embodiment of the inventionmay further comprise a package insert indicating that the compositionscan be used to treat a particular condition. Alternatively, oradditionally, the article of manufacture or kit may further comprise asecond (or third) container comprising a pharmaceutically-acceptablebuffer, such as bacteriostatic water for injection (BWFI),phosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles, andsyringes.

In some embodiments, provided herein is a kit comprising a medicamentcomprising an anti-angiogenesis agent and an optional pharmaceuticallyacceptable carrier, and a package insert comprising instructions foradministration of the medicament in combination with a compositioncomprising an OX40 binding agonist and an optional pharmaceuticallyacceptable carrier for treating or delaying progression of cancer in anindividual. Further provided here is a kit comprising a first medicamentcomprising an anti-angiogenesis agent and an optional pharmaceuticallyacceptable carrier, and a second medicament comprising an OX40 bindingagonist and an optional pharmaceutically acceptable carrier. In someembodiments, the kit further comprises a package insert comprisinginstructions for administration of the first medicament and the secondmedicament for treating or delaying progression of cancer in anindividual. Still further provided herein is a kit comprising amedicament comprising an OX40 binding agonist and an optionalpharmaceutically acceptable carrier, and a package insert comprisinginstructions for administration of the medicament in combination with acomposition comprising an anti-angiogenesis agent and an optionalpharmaceutically acceptable carrier for treating or delaying progressionof cancer in an individual.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-OX40 antibody and/or anti-angiogenesis agent.

The specification is considered to be sufficient to enable one skilledin the art to practice the invention. Various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims.

Example 1: Combinatorial Anti-VEGF+Anti-OX40 Treatment Exhibited GreaterEfficacy than Single Agent Treatment

Combining different modalities for cancer treatment may result inbeneficial effects on tumor growth. As described below, anti-VEGFtreatment resulted in reduced tumor growth when compared to anti-GP120(control) treated mice. Anti-OX40 treatment exhibited little activityalone. Surprisingly, despite little anti-tumor activity on its own,anti-OX40 treatment in combination with anti-VEGF treatment demonstratedsuperior tumor growth inhibition when compared to single agentadministration or anti-GP120 treatment. Without wishing to be bound totheory, it is hypothesized that the synergistic enhancement of activityobserved with combinatorial anti-VEGF+anti-OX40 treatment may be due toincreased intratumoral dendritic cell activation induced by anti-VEGFtreatment.

The results described herein suggest that sequencing anti-OX40 treatmentwith anti-VEGF administration may augment therapy. For instance, withoutwishing to be bound to theory, administering anti-VEGF first (therebyenhancing dendritic cell activation), followed by anti-OX40 therapy, maybe more effective than co-administration of treatments. However, it ispossible that the anti-angiogenic effects of anti-VEGF treatment maydeplete vasculature, thereby limiting leukocyte infiltration. Therefore,without wishing to be bound to theory, it may be more beneficial toadminister anti-OX40 prior to anti-VEGF treatment.

Materials and Methods

CT26Mouse Tumor Model

6 week old female Balb/C mice were inoculated subcutaneously in theright hind flank with 100 μl of HBSS+matrigel (according tomanufacturer's specifications) containing 1×10⁵ CT26 tumor cells. Tumorswere allowed to grow for −2 weeks. Mice were grouped into four differentexperimental arms of 10 mice per arm. Groups were selected to havesimilar tumor volume averages with a tumor volume range from 100-300mm³.

Mice received 0.1 mg/kg of anti-OX40 or isotype control anti-GP 120(negative control). The anti-OX40 antibody was clone OX-86 mouse-IgG2a(generated by cloning rat anti-mouse OX40 agonist antibody OX-86 onto amurine IgG2a backbone). Antibody was administered intravenously on day 1followed by 0.1 mg/kg of the same antibody intraperitoneally (i.p.) 3times a week for a total treatment duration of three weeks. These sametreatment groups also received 5 mg/kg of anti-VEGFA (B20) or isotypecontrol anti-GP120 (negative control) i.p. twice a week for three weeksstarting on day 1.

Antibodies were diluted to the desired concentration with sterile PBSand administered in volumes of 100 or 200 μl. Tumor volume was measuredby calipers periodically over the duration of the experiment. Mice wereeuthanized and removed from the experiment when: 1) Mice becamemoribund, 2) Tumor became ulcerated, or 3) Tumor volume exceeded 2000mm³.

Flow Cytometry

CT26 tumors harvested from mice treated with anti-VEGF or anti-GP120(control) were subjected to enzymatic digestion for retrieval of asuspension of single cells. Subsequently, these cells were stained forflow cytometry using a cocktail of antibodies against CD45, CD11b,CD11c, F4/80 (used for exclusion of macrophages), Gr1 (used forexclusion of neutrophils and granulocytic myeloid cells), MHC-II, OX40L,and PD-L1. Fixable Viability Dye eFluor® 780 was used for exclusion ofdead cells from flow cytometric analysis. Myeloid dendritic cells weredefined and gated as CD45⁺CD11b⁺Gr1⁻ F4/80⁻CD11c⁺MHCII⁺. Non-myeloiddendritic cells were defined and gated as CD45⁺CD11b⁻Gr1⁻F4/80⁻CD11c⁺MHCII⁺. Expression of the functional markers MHCII, PD-L1,and OX40L was quantified by means of flow cytometric Mean FluorescenceIntensity using the following antibodies: PeCy7-conjugated anti-MHC-II(Biolegend), BV421-conjugated anti-PD-L1 (Biolegend), and PE-conjugatedOX-40L.

Results

To determine the effect of combination treatment with anti-OX40 andanti-VEGF on tumor growth, a mouse CT26 tumor model was used. FIG. 1shows that anti-VEGF treatment plus isotype control inhibited tumorgrowth when compared to anti-GP120 negative control administration. Incontrast, anti-OX40 plus isotype control afforded no inhibition of tumorgrowth when compared to anti-GP120 negative control administration thisexperiment. This is inconsistent with other experiments utilizing thesame antibody and experimental tumor model where tumor growth inhibitionwas observed. Without wishing to be bound to theory, an explanation forwhy anti-OX40 treatment did not work in this experiment is that theaverage starting tumor volume was larger in this group and anti-OX40efficacy in the CT26 tumor model is dramatically affected by tumor sizeespecially when tumors are larger than 200 mm³. It is thought thatanti-OX40 efficacy in the CT26 tumor model may be negatively affected bytumor size. FIG. 1 also shows that combinatorial anti-VEGF and anti-OX40treatment showed superior efficacy over anti-VEGF or anti-OX40 alone.

FIG. 2 provides tumor volume measurements for individual mice. Thesedata further demonstrate the superior efficacy of combinatorialanti-VEGF and anti-OX40 treatment over anti-VEGF or anti-OX40 alone.Compared to tumor growth in mice receiving control treatment, micereceiving VEGF treatment experienced 53% tumor growth inhibition.Anti-OX40 treatment alone resulted in tumor growth 30% above controltreatment. In contrast, combination anti-VEGF plus anti-OX40 treatmentresulted in a remarkable 94% tumor growth inhibition compared to controltreatment. In this treatment group, 9 out of 10 mice exhibited tumorstasis or regression. These results demonstrate the superior andsynergistic effects of combination anti-VEGF and anti-OX40 treatment, ascompared to each single treatment or control treatment.

Next, the effect of anti-VEGF treatment on intratumoral dendritic cellactivation was tested in the CT26 tumor model. First, in FIG. 3A,intratumoral myeloid dendritic cells were assayed. AmongCD45⁺CD11b⁺Gr1⁻F4/80⁻CD11c⁺MHCII⁺ myeloid dendritic cells, abundance ofexpression of MHC class II, OX40L, and PD-L1 was determined byquantifying the mean fluorescence intensity of each molecule. Myeloiddendritic cells from anti-VEGF-treated mice were found to have higherMHC II (p=0.002) and OX40L (p=0.003) expression, as compared to cellsfrom anti-GP 120 (control)-treated mice. PD-L1 expression, a negativeregulator of T cell responses, was statistically undistinguishablebetween the two groups (p=0.81). These results suggest that treatmentwith anti-VEGF promoted maturation of tumoral dendritic cells as opposedto control treatment. Improved expression of MHC Class II and OX40Lenables dendritic cells to present antigens and prime T cells moreeffectively.

FIG. 3B shows the effect of anti-VEGF treatment on non-myeloidintratumoral dendritic cell activation. Among CD45+CD11b⁻Gr1⁻F4/80⁻CD11c⁺MHCII⁺ non-myeloid dendritic cells, expression of MHC classII, as well as PD-L1 and OX40L was determined by quantifying the meanfluorescence intensity of each molecule. Similar to myeloid dendriticcells, non-myeloid dendritic cells also expressed significantly higherlevels of MHC II (p=0.03) and OX40L (p=0.002) when treated withanti-VEGF, as compared to control treatment.

These results suggest that anti-VEGF treatment can improve thefunctional phenotype of tumoral dendritic cells, a phenomenon that canresult in enhanced anti-tumoral T cell responses. Hence, combininganti-VEGF treatment with immunotherapeutics targeting T cells (e.g.,anti-OX40) may synergistically potentiate anti-tumor responses.

1. A method for treating or delaying progression of cancer in anindividual comprising administering to the individual an effectiveamount of an anti-angiogenesis agent and an OX40 binding agonist.
 2. Themethod of claim 1, wherein the anti-angiogenesis agent is selected fromthe group consisting of an anti-VEGFR2 antibody; an anti-VEGFR1antibody; a VEGF-trap; a bispecific VEGF antibody; a bispecific antibodycomprising a combination of two arms selected from the group consistingof an anti-VEGF arm, an anti-VEGFR1 arm, and an anti-VEGFR2 arm; ananti-VEGF-A antibody; an anti-VEGFB antibody; an anti-VEGFC antibody; ananti-VEGFD antibody; a nonpeptide small molecule VEGF antagonist; ananti-PDGFR inhibitor; and a native angiogenesis inhibitor.
 3. The methodof claim 2, wherein the anti-angiogenesis agent is selected from thegroup consisting of ramucirumab, tanibirumab, aflibercept, icrucumab,ziv-aflibercept, MP-0250, vanucizumab, sevacizumab, VGX-100, pazopanib,axitinib, vandetanib, stivarga, cabozantinib, lenvatinib, nintedanib,orantinib, telatinib, dovitinig, cediranib, motesanib, sulfatinib,apatinib, foretinib, famitinib, imatinib, and tivozanib.
 4. The methodof claim 1, wherein the anti-angiogenesis agent is an anti-angiogenesisantibody.
 5. The method of claim 4, wherein the anti-angiogenesisantibody is a monoclonal antibody.
 6. The method of claim 4, wherein theanti-angiogenesis antibody is a human or humanized antibody.
 7. Themethod of claim 1, wherein the anti-angiogenesis agent is a VEGFantagonist.
 8. The method of claim 7, wherein the VEGF antagonistreduces the expression level or biological activity of VEGF by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
 9. The method of claim8, wherein the VEGF is VEGF (8-109), VEGF (1-109), or VEGF₁₆₅.
 10. Themethod of claim 7, wherein the VEGF antagonist increases MHC class IIexpression on dendritic cells as compared to MHC class II expression ondendritic cells prior to treatment with the VEGF antagonist.
 11. Themethod of claim 7, wherein the VEGF antagonist increases OX40Lexpression on dendritic cells as compared to OX40L expression ondendritic cells prior to treatment with the VEGF antagonist.
 12. Themethod of claim 11, wherein the dendritic cells are myeloid dendriticcells.
 13. The method of claim 11, wherein the dendritic cells arenon-myeloid dendritic cells.
 14. The method of claim 7, wherein the VEGFantagonist comprises a soluble VEGF receptor or a soluble VEGF receptorfragment that specifically binds to VEGF.
 15. The method of claim 7,wherein the VEGF antagonist is a chimeric VEGF receptor protein.
 16. Themethod of claim 7, wherein the VEGF antagonist is administered by genetherapy.
 17. The method of claim 7, wherein the VEGF antagonist is ananti-VEGF antibody.
 18. The method of claim 17, wherein the anti-VEGFantibody is a human or humanized antibody.
 19. The method of claim 17,wherein the anti-VEGF antibody binds to the A4.6.1 epitope.
 20. Themethod of claim 17, wherein the anti-VEGF antibody binds to a functionalepitope comprising residues F17, M18, D19, Y21, Y25, Q89, 191, K101,E103, and C104 of human VEGF.
 21. The method of claim 17, wherein theanti-VEGF antibody binds to a functional epitope comprising residuesF17, Y21, Q22, Y25, D63, 183, and Q89 of human VEGF.
 22. The method ofclaim 17, wherein the anti-VEGF antibody is a G6 series antibody. 23.The method of claim 17, wherein the anti-VEGF antibody is a B20 seriesantibody.
 24. The method of claim 17, wherein the anti-VEGF antibody isa monoclonal anti-VEGF antibody.
 25. The method of claim 24, wherein themonoclonal anti-VEGF antibody is bevacizumab.
 26. The method of claim17, wherein the anti-VEGF antibody comprises a light chain variableregion comprising the amino acid sequence of DIQMTQSPSS LSASVGDRVTITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ YSTVPWTFGQ GTKVEIKR. (SEQ ID NO:214).
 27. The method of claim17, wherein the anti-VEGF antibody comprises a heavy chain variableregion comprising the amino acid sequence of EVQLVESGGG LVQPGGSLRLSCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAYLQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS (SEQ ID NO:215).
 28. Themethod of claim 17, wherein the anti-VEGF antibody comprises a lightchain variable region comprising the amino acid sequence of DIQMTQSPSSLSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTDFTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKR. (SEQ ID NO:214) and a heavychain variable region comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTFSLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS (SEQ IDNO:215).
 29. The method of claim 17, wherein the anti-VEGF antibodycomprises one, two, three, four, five, or six hypervariable region (HVR)sequences of bevacizumab.
 30. The method of claim 1, wherein the OX40binding agonist is selected from the group consisting of an OX40 agonistantibody, an OX40L agonist fragment, an OX40 oligomeric receptor, and anOX40 immunoadhesin.
 31. The method of claim 1, wherein the OX40 bindingagonist is a trimeric OX40L-Fc protein.
 32. The method of claim 1,wherein the OX40 binding agonist is an OX40L agonist fragment comprisingone or more extracellular domains of OX40L.
 33. The method of claim 1,wherein the OX40 binding agonist is an OX40 agonist antibody that bindshuman OX40.
 34. The method of claim 33, wherein the OX40 agonistantibody is a full-length human IgG1 antibody.
 35. The method of claim33, wherein the OX40 agonist antibody depletes cells that express humanOX40.
 36. The method of claim 35, wherein the cells are CD4+ effector Tcells.
 37. The method of claim 35, wherein the cells are Treg cells. 38.The method of claim 35, wherein the depleting is by ADCC and/orphagocytosis.
 39. The method of claim 38, wherein the depleting is byADCC.
 40. The method of claim 33, wherein the OX40 agonist antibodybinds human OX40 with an affinity of less than or equal to about 0.45nM.
 41. The method of claim 40, wherein the OX40 agonist antibody bindshuman OX40 with an affinity of less than or equal to about 0.4 nM. 42.The method of claim 40, wherein OX40 agonist antibody binding affinityis determined using radioimmunoassay.
 43. The method of claim 33,wherein binding to human OX40 has an EC50 of less than or equal to 0.2ug/ml.
 44. The method of claim 33, wherein binding to human OX40 has anEC50 of less than or equal to 0.3 ug/ml.
 45. The method of claim 33,wherein the OX40 agonist antibody increases CD4+ effector T cellproliferation and/or increasing cytokine production by the CD4+ effectorT cell as compared to proliferation and/or cytokine production prior totreatment with anti-human OX40 agonist antibody.
 46. The method of claim45, wherein the cytokine is gamma interferon.
 47. The method of claim33, wherein the OX40 agonist antibody increases memory T cellproliferation and/or increasing cytokine production by the memory cell.48. The method of claim 47, wherein the cytokine is gamma interferon.49. The method of claim 33, wherein the OX40 agonist antibody inhibitsTreg function.
 50. The method of claim 49, wherein the OX40 agonistantibody inhibits Treg suppression of effector T cell function.
 51. Themethod of claim 50, wherein effector T cell function is effector T cellproliferation and/or cytokine production.
 52. The method of claim 50,wherein the effector T cell is a CD4+ effector T cell.
 53. The method ofclaim 33, wherein the OX40 agonist antibody increases OX40 signaltransduction in a target cell that expresses OX40.
 54. The method ofclaim 53, wherein OX40 signal transduction is detected by monitoringNFkB downstream signaling.
 55. The method of claim 33, wherein the OX40agonist antibody is stable after treatment at 40° C. for two weeks. 56.The method of claim 33, wherein the OX40 agonist antibody comprises avariant IgG1 Fc polypeptide comprising a mutation that eliminatesbinding to human effector cells, and wherein the antibody has diminishedactivity relative to an anti-human OX40 agonist antibody comprising anative sequence IgG1 Fc portion.
 57. The method of claim 56, wherein theOX40 agonist antibody comprises a variant Fc portion comprising a DANAmutation.
 58. The method of claim 33, wherein OX40 agonist antibodycross-linking is required for anti-human OX40 agonist antibody function.59. The method of claim 33, the OX40 agonist antibody comprises (a) a VHdomain comprising (i) HVR-H1 comprising the amino acid sequence of SEQID NO: 2, 8 or 9, (ii) HVR-H2 comprising the amino acid sequence of SEQID NO: 3, 10, 11, 12, 13 or 14, and (iii) HVR-H3 comprising an aminoacid sequence selected from SEQ ID NO: 4, 15, or 19; and (iv) HVR-L1comprising the amino acid sequence of SEQ ID NO:5, (v) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6, and (vi) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 7, 22, 23, 24, 25, 26, 27, or
 28. 60.The method of claim 59, wherein the OX40 agonist antibody comprises (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:2; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:3; (c) HVR-H3 comprisingthe amino acid sequence of SEQ ID NO:4; (d) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acidsequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acidsequence of SEQ ID NO:7.
 61. The method of claim 59, wherein the OX40agonist antibody comprises (a) HVR-H1 comprising the amino acid sequenceof SEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3comprising an amino acid sequence of SEQ ID NO:26.
 62. The method ofclaim 59, wherein the OX40 agonist antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprisingthe amino acid sequence of SEQ ID NO:3; (c) HVR-H3 comprising the aminoacid sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acidsequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequenceof SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid sequence of SEQID NO:27.
 63. The method of claim 33, wherein the OX40 agonist antibodycomprises a heavy chain variable domain (VH) sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,108, 114, 116, 233, or
 234. 64. The method of claim 33, wherein the OX40agonist antibody comprises a light chain variable domain (VL) having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,109, 115 or
 117. 65. The method of claim 33, wherein the OX40 agonistantibody comprises a heavy chain variable domain (VH) sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:56.
 66. The method ofclaim 65, wherein the OX40 agonist VH sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions, insertions, or deletions relative to the referencesequence, but an anti-human OX40 agonist antibody comprising thatsequence retains the ability to bind to human OX40.
 67. The method ofclaim 65, wherein a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO:56.
 68. The method of claim 65,wherein the OX40 agonist VH comprises one, two or three HVRs selectedfrom: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:4.
 69. The method ofclaim 33, wherein the OX40 agonist antibody comprises a light chainvariable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID NO:57.
 70. The method of claim 69, wherein the OX40 agonist VLsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions, insertions, or deletions relativeto the reference sequence, but an anti-human OX40 agonist antibodycomprising that sequence retains the ability to bind to human OX40. 71.The method of claim 69, wherein a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO:
 57. 72. The method ofclaim 69, wherein the OX40 agonist VL comprises one, two or three HVRsselected from (a) HVR-L1 comprising the amino acid sequence of SEQ IDNO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.
 73. Themethod of claim 33, wherein the OX40 agonist antibody comprises a VHsequence of SEQ ID NO:
 56. 74. The method of claim 33, wherein the OX40agonist antibody comprises a VL sequence of SEQ ID NO:
 57. 75. Themethod of claim 33, wherein the OX40 agonist antibody comprises a VHsequence of SEQ ID NO:56 and a VL sequence of SEQ ID NO:
 57. 76. Themethod of claim 33, wherein the OX40 agonist antibody comprises a VHsequence of SEQ ID NO:
 94. 77. The method of claim 33, wherein the OX40agonist antibody comprises a VL sequence of SEQ ID NO:
 95. 78. Themethod of claim 33, wherein the OX40 agonist antibody comprises a VHsequence of SEQ ID NO:94 and a VL sequence of SEQ ID NO:
 95. 79. Themethod of claim 33, wherein the OX40 agonist antibody comprises a VHsequence of SEQ ID NO:
 96. 80. The method of claim 33, wherein the OX40agonist antibody comprises a VL sequence of SEQ ID NO:
 97. 81. Themethod of claim 33, wherein the OX40 agonist antibody comprises a VHsequence of SEQ ID NO:96 and a VL sequence of SEQ ID NO:
 97. 82. Themethod of claim 33, wherein the OX40 agonist antibody is MEDI6469,MEDI0562, or MEDI6383.
 83. The method of claim 1, wherein the cancer islung cancer, glioblastoma, cervical cancer, ovarian cancer, breastcancer, colon cancer, colorectal cancer, fallopian tube cancer,peritoneal cancer, kidney cancer, renal cancer, non-Hodgkins lymphoma,prostate cancer, pancreatic cancer, soft-tissue sarcoma, kaposi'ssarcoma, carcinoid carcinoma, head and neck cancer, mesothelioma,multiple myeloma, non-small cell lung cancer, neuroblastoma, melanoma,gastric cancer, or liver cancer.
 84. The method of claim 1, wherein thecancer is a gynecologic cancer.
 85. The method of claim 1, wherein thecancer is advanced, refractory, recurrent, chemotherapy-resistant,and/or platinum-resistant.
 86. The method of claim 1, wherein theindividual has cancer or has been diagnosed with cancer.
 87. The methodof claim 1, wherein the treatment results in a sustained response in theindividual after cessation of the treatment.
 88. The method of claim 1,wherein the OX40 binding agonist is administered before theanti-angiogenesis agent, simultaneous with the anti-angiogenesis agent,or after the anti-angiogenesis agent.
 89. The method of claim 1, whereinthe individual is a human.
 90. The method of claim 1, wherein theanti-angiogenesis agent and/or the OX40 binding agonist are administeredintravenously, intramuscularly, subcutaneously, intracerobrospinally,topically, orally, transdermally, intraperitoneally, intraorbitally, byimplantation, by inhalation, intrathecally, intraventricularly,intra-articularly, intrasynovially, or intranasally.
 91. The method ofclaim 1, further comprising administering a chemotherapeutic agent fortreating or delaying progression of cancer. 92-95. (canceled)
 96. A kitcomprising a medicament comprising an anti-angiogenesis agent and anoptional pharmaceutically acceptable carrier, and a package insertcomprising instructions for administration of the medicament incombination with a composition comprising an OX40 binding agonist and anoptional pharmaceutically acceptable carrier for treating or delayingprogression of cancer in an individual.
 97. A kit comprising a firstmedicament comprising an anti-angiogenesis agent and an optionalpharmaceutically acceptable carrier, and a second medicament comprisingan OX40 binding agonist and an optional pharmaceutically acceptablecarrier.
 98. The kit of claim 97, wherein the kit further comprises apackage insert comprising instructions for administration of the firstmedicament and the second medicament for treating or delayingprogression of cancer in an individual.
 99. A kit comprising amedicament comprising an OX40 binding agonist and an optionalpharmaceutically acceptable carrier, and a package insert comprisinginstructions for administration of the medicament in combination with acomposition comprising an anti-angiogenesis agent and an optionalpharmaceutically acceptable carrier for treating or delaying progressionof cancer in an individual.